이질아메바 (Entamoeba histolytica)의 주별(株別)병원성에 관한 실험적 연구
Experimental studies on the pathogenicity of entamoba histolytica strains
Until Neal's successful reports (1951) utilizing Wistar strain rats, rats had been regarded as unsuitable hosts in the studies of infections of Entamoeba histolytica. After Thompson et al. (1953) did a comparative virulence study of two strains of Entamoeba histolytica using Sprague-Dawley strain rats, the experimental infection of rats with Entamoeba histolytica was renewed. It opened a new line of inquiry into some of the problems of amebiasis (vincent and Neal, 1960; Bird and Neal, 1963; Singh et al., 1963; Kradolfer and Jarumilinta, 1965; Healy and Gleason, 1966; Neal et al., 1968; Neal and Johson, 1968).
The present observations deal with a comparison of the virulence of Entamoeba histolytica to the Sprague-Dawley strain rats according to animal ages, number of inoculated amebae, rat strain differences and rat-culture passages. The cecal scoring technique by Neal (1951) was applied.
Strains of Entamoeba histolytica:
Seven strains of Entamoeba histolytica have been studied.
1) YS9-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) NAMRU Ⅱ-strain. The strain, obtained from NAMRU No. 2(Taiwan) through the courtesy of Dr. J. H. Cross, was isolated in 1967 from an acute amebic dysentery patient (Vietnam dweller) by rectoscopic method, and subcultured.
3) YS14-strain. The ameba was isolated in January 1967 from a 63-year-old healthy cyst-passer's stool (man, Cheju islander), and maintained by subculture.
4) YS15-strain. The strain was collected in January 1968 from the feces of a 51-year-old symptomless cyst carrier (man, Suwon inhabitant), and maintained as above.
5) Ys16-strain. This was isolated in February 1968 from a 45-year-old woman, healthy cyst-passer, at Severance Hospital, and subcultured.
6) YS24-strain and 7) YS25-strain. These strains were collected in June 1969 from trophozoites in the liver abscess of a 33, and a 42-year-old man respectively at Severance Hospital and associated with the bacterial flora of NAMRU Ⅱ-strain ameba, and maintained as above.
Sprague-Dawley strain and hybrid rat were used. The animals were inoculated with Entamoeba histolytica intracecally following the technique described by Jones (1946). The rats were killed by ether anesthesia one week after inoculation and the entire cecum was removed and slit opened. The condition of the cecal contents were recorded by the procedure of Neal (1951).
The following are the criteria for scoring used.
Contents: Normal 0
Slightly less solid than normal 1
Slightly mucoid 2
Mucoid, some solid matter present 3
NO solid matter, white or yellow mucus only 4
Wall : Normal 0
Slight thickening 1
Marked local thickening and contraction 2
Extensive thickening and contraction 3
Cecum shapeless, extensive ulceration with abscess fromation 4
The average cecal score was calculated only from infected rats.
Rat-culture passage was conducted in order to investigate loss or increase of invasiveness of the organisms according to amoeba strains, or concomitant bacterial flora differences.
For the experiment on invasiveness of Entamoeba histolytica according to the host ages, YS9 and NAMRU Ⅱ-strain amebae were applied to Sprague-Dawley strain rat. Each animal was inoculated equally with 100,000 organisms. In the YS9-strain ameba infected rats, four groups wre compared; 30-day-old (Group A^^1 , A^^2), 40-day-old (Group B^^1 , B^^2), 60-day-old (Group C) and 90-day-old (Group D). Average cecal scores showed 7.8±0.10 in Group A^^1, 50.±0.43 in Group A^^2 , 4.2±0.48 in Group B^^1 , 3.4±0.22 in Group B^^2 , 2.5±0.31 in Group C and 2.8±0.32 in Group D. It indicated a high degree of invasiveness in Group A.
In the study of NAMRU Ⅱ-strain ameba inoculated rats, three groups were compared; 20-day-old (Group A), 30-day-old (Group B) and 50-day-old (Group C^^1 , C^^2 , C^^3). Average cecal scores showed 3.4±0.29 in Group A, 5.6±0.36 in Group B, 2.0±0.00 in Group C^^1 , 1.0±0.40 in Group C^^2 and 0.2±0.95 in Group C^^3.
A high degree of invasiveness was observed in Group B.
In the experiment on virulence of Entamoeba histolytica in relation to the number of inoculated amebae, the animals were of fully weaned Sprague-Dawley strain rats, which were 30-day-old and 30-40 grams in weigh, and the ameba was of YS9-strain. The rats were inoculated 50,000 organisms in group B, 200,000 in Group C and 500,000 in Group D^^1 and D^^2. Average cecal scores showed 3.3±0.66 in Group B, 6.0±0.38 in Group C, 7.2±0.25 in Group D^^1 and 3.2±0.63 in Group D^^2. Comparing the results with Group A, which was inoculated with 100,000 amebase as in
the preceding experiment, the average cecal scores showed no statistical differences in Group A, C and D, and a high degree of invasiveness was indicated since it had moer than 5.0 of the average cecal score, but marked difference was observed in Group B.
In ;the study on invasiveness of YS9-strain ameba using hyvrid of albino rats, the animals were fully weaned. Each group was composed of the same mother-born rats, and each animal was inoculated equally with 100,000 organisms. The experiment was performed in 6 groups. Average cecal scores showed 6.4±0.25 in Group 1, 0.7±0.21 in Group 2, 2.4±0.49 in Group 3, 3.3±0.65 in Group 4, 6.0±0.66 in Group 5 and 2.5±0.95 in Group 6. Generally, as the results with hybrid of alvino rats were conflicting, the animal was to be considered unsuitable for the study on virulence of Entamoeba histolytica.
In the experiment on virulence regarding ameba strains, the rats were of the sprague-Dawley strain, and each group was composed of the same mother-born rats. Each animal was inoculated with 100,000∼200,000 amebae. YS14, 15 and 16-strain amebae, orginating from cyst carriers, YS24 and 25-strain amebae, isolated from liver abscess, were used. Average cecal scores showed 1.0±0.22 at the YS14-strain ameba inoculated group, 3.0±0.38 at the YS15-strain ameba group, 2.2±0.28 at the YS16-strain ameba grup, 3.0±1.73 at YS24-strain group and 6.3±0.75 at YS25-strain group. Comparing these results with YS9-strain and NAMRU Ⅱ-strain amebae, YS14 and YS16-strain showed no virulence; YS15 and YS24-strain were moderately invasive, and only YS25-strain was determined as a highly invasive strain.
In order to study the virulent differences of ameba strains between the pathological results in animal and clinical manifestation, the rat-culture passage experiments were carried out. Liver abscess born YS24-strain ameba, which shoved 3.0±1.73 with average cecal score first, the virulence increased to 4.0±0.00 at second rat passage. Among the cyst carrier born amebae, YS14-strain showed marked increase on virulence with average cecal score 5.5±0.90 at second rat passage, but no changes were observed in other strains. On the other hands, YS9, NAMRU Ⅱ and YS25-strains showed no difference and continued their virulences with average cecal scored more than 5.0 by rat-culture passages.
The experimental infections of rats with 7 strains of Entamoeba histolytic were carried out, and the rat cecal scoring technique of Neal (1951) was utilized to measure the invasiveness of the parasites.
1) IN the infection of Sprague-Dawley strain rat with YS9-strain and NAMRU Ⅱ-strain amebae, remarkable invasiveness was observed in the 30-day-old rat groups with the average cecal scored above 5.0.
Although no statistical differences os virulence by the number of inoculations showed up in the rat groups, cecal scores were markedly reduced in the 50,000 amebae inoculated rats.
2) The hybrid of albino rats were considered unsuitable for the study for virulence of Entamoeba histolytica, since the invasiveness of the amebae gave inconsistant results in every experiment, it was difficult to interpret the virulency.
3) In the virulence regarding to ameba strains, however YS14, YS15 and YS16-strains were originated from cyst carriers, and YS24 and YS25-strains were isolated from liver abscess, YS14 and YS16-strains showed no virulence, YS15 and YS24-strains were moderately invasive, and only YS25-strains was observed as a
highly invasive strain compared with YS9 and NAMRU Ⅱ-strain amebae. By rat-culture passage, YS14-strain and YS24-strain amebae showed marked increase of invasiveness.
It was presumed that the rat-culture passage should be indispensably supplemented in the studies on the virulence of Entamoeba histolytica.