Experimental studies on the of protein deficiency on the infectivity of entamoeba histolytica

의학과/박사[한글] Experimental Studies on the Effect of Protein Deficiency on the Infectivity of Entamoeba histolytica Kil-Choon Choi, M.D. Department of parasitology Graduate School, Yonsei University (Directors: Prof. Chin-Thack Soh, Assoc. Prof. Keun-Tae Lee) Introduction There have been a few reports dealing with the relationship between the nutritional condition of the host and parasitic infestation. Taylor(1952) reported improvement in the clinical manifestations of disease and decreased worm incidence in the host which and received an adequate amount of animal protein. However, Krebs(1946) found that the patients having hypoproteinemia due to the malnutrition showed a decreased γ-globulin level when compared to the control group fed hyperprotien diets. Recently, Clarke91968) reported that more larvae of Nippostrongylus braziliensis migrated to the lungs and intestine in rats feds a low protein diet than in the control diet group. In regard to the infection due to Entamoeba histolytica, Taylor(1950), Sedun et al.(1951) suggested that the intensity was correlated with the type of diet. However there has been no report concerning the relationship of the nutritional condition of the host and infectivity of E. histolytica. The present investigation was designed to study rat susceptibility or resistance to E. histolytica infection under varying levels of dietary proteins. Materials and Methods 1. Animals: Young rats of both sexes were used. Animals were about 40 days old and weighed 35 to 40 grams. Each rat was isolated and fed with the designed diet. Body weight was checked every other day. 2. Strain of E. histolytic: YS-9 was isolated from Korean patient and stabilized by successive subcultures. 3. Diets : The experimental diets divided into 4 groups are as follows: 1) Group D ; Depleted protein diet(rise powder 85% and no casein) 2) Group L ; Low protein diet(rice powder 80%, casein 5%) 3) Group M ; Moderate protein diet(rice powder 70%, casein 5%) 4) Group H ; High protein diet(rice powder 60%, casein 25%). Olive oil(4%). inorganic salt mixture(4%), cod liver oil(2%) and yeast(5%)were added in each diet with the same rate. (Salt mixture was made by the prescription of H.M. Wakeman, 1937) 4. Animal grouping of 90 rats were divided into 4 groups: 30 rats in group M and 20 rats each in groups D, L and H. Forty to 80gms diet was supplied to all rats every other day. The ingested and residual volumes of the diet were checked. The weight of the rats was measured from the day before the initiation of experimental feeding up to the date for the experiment to be finished. 5. Inoculation technique: E. histolytica was inoculated into the experimental rats which were fed by the experimental diets for 15 to 17 days. Amoebae were pooled from 48-hour-old culture tubes and spun for 5 minutes at 800-1,000 rpm. The sedimented amoebae were suspended in sterile saline containing 200,000 organisms per ml. The rats were anesthetized with ether, an incision was made slightly to the left of midline of the abdomen, and the cecum partially exposed. Through a 5ml. syringe and a 23 gauge needle, the inoculum was injected toward the blind end of the cecum form a point anterior to the junction of the cecum and colon. The abdominal muscles and skin were then sutured with the surgical silk. 6. Paper electrophoresis of the rat serum protein: Rat sera were obtained from the cut tail using heparinized capilary tubes. For electrophoresis studies the sera were then placed in the Spinco electrophoresis chamber(Beckman Co.) on cellulose polyacetate strips in veronal buffer solution 9pH; 8.6) Each strip was stained in Ponceu S solution for 30 minutes. Thereafter serim protein fractionation was carried out by the elurion techique. The optical density of each fraction was calculated in the spectrophotometer at light length of 540mμ. 7. Identification of the pathogenicity of E. histolytica in experimental rats: The rats were sacrificed with ether 14 days after inoculation of E. histolytica. The entire cecum was removed and opened. The cecal content was examined for the presence of amoebae in a direct saline wet mount. The number and size of the crater-like ulcers in the ileoceal area were measured under the microscope, and then histo-pathological studies were carried on. The ulcers were divided into 3 degrees of severity of the ulceration as follows: Degree Ⅰ : One pin point ulcer in ileocecal area of rat. Degree Ⅱ : One to 2 ulcers of 1-2 mm in diameter Degree Ⅲ : More than 2 ulcers. Results and summary 1. The growth rates of rats according to the various diets. The growth of the depleted protein diet group(D) was markedly reduced more than that of any other group from 3rd day of diet control. The amount of ingested diet in the various groups did not show any difference statistically. An average amount consumed was 30-40gms day per capita. 2. Detection rate of E. histolytica trophozoites in each group. All the experimental rats were sacrificed 14th day after the inoculation with E.histolytica. Amoebae were found from the contents in the ileocecal area of the rats as follows: 100% in group D; 85.7% in group L; 736% in group M; and 44.4% in group H. 3. Electrophoretic fractionation of serum protein in rats. The serum protein fraction in each group before and after inoculation of E. histolytica is shown in the following table. Generally the γ-globulin level was increased in all the groups after the inoculation of amoebae, specially in hyperprotein diet group. 4. Total serum protein. The value of total serum protein in Group D (6.87gm%) was the lowest in any group. 5. Histological findings. ---------+---------------------------------+-----------------------------------A/G ratio4.1 ---------+----------------+----------------+----------------------------------- The percentage of the ulceration was 60.0% in group D and 21.4% in group L. In group M and H. each had only one case showing cecal ulceration. Conclusion Parasitological examination of the ileocecal area and serological studies were made on rats fec ranging levels of protein and them infected with E. histolytica. Rat fed deplete or lower protein diets had a higher incidence of ilocecal contents positive for amoeba. The cecal ulceration rate and the infectivity of E. histolytica were increased in rats on a low protein diet as compared with rats fed a high protein diet. Although γ-globulin level in the sera of all the rats was increased after the inoculation of E. histolytica, the level was markedly elevated in the rats on the high protein diet. Through above results, it is summarized hat the less doses of protein diet retard the growth of host and decrease the resistance to amoebic infection.4.1 ---------+----------------+----------------+----------------------------------- The percentage of the ulceration was 60.0% in group D and 21.4% in group L. In group M and H. each had only one case showing cecal ulceration. Conclusion Parasitological examination of the ileocecal area and serological studies were made on rats fec ranging levels of protein and them infected with E. histolytica. Rat fed deplete or lower protein diets had a higher incidence of ilocecal contents positive for amoeba. The cecal ulceration rate and the infectivity of E. histolytica were increased in rats on a low protein diet as compared with rats fed a high protein diet. Although γ-globulin level in the sera of all the rats was increased after the inoculation of E. histolytica, the level was markedly elevated in the rats on the high protein diet. Through above results, it is summarized hat the less doses of protein diet retard the growth of host and decrease the resistance to amoebic infection. [영문] There have been a few reports dealing with the relationship between the nutritional condition of the host and parasitic infestation. Taylor(1952) reported improvement in the clinical manifestations of disease and decreased worm incidence in the host which and received an adequate amount of animal protein. However, Krebs(1946) found that the patients having hypoproteinemia due to the malnutrition showed a decreased γ-globulin level when compared to the control group fed hyperprotien diets. Recently, Clarke91968) reported that more larvae of Nippostrongylus braziliensis migrated to the lungs and intestine in rats feds a low protein diet than in the control diet group. In regard to the infection due to Entamoeba histolytica, Taylor(1950), Sedun et al.(1951) suggested that the intensity was correlated with the type of diet. However there has been no report concerning the relationship of the nutritional condition of the host and infectivity of E. histolytica. The present investigation was designed to study rat susceptibility or resistance to E. histolytica infection under varying levels of dietary proteins. Materials and Methods 1. Animals: Young rats of both sexes were used. Animals were about 40 days old and weighed 35 to 40 grams. Each rat was isolated and fed with the designed diet. Body weight was checked every other day. 2. Strain of E. histolytic: YS-9 was isolated from Korean patient and stabilized by successive subcultures. 3. Diets : The experimental diets divided into 4 groups are as follows: 1) Group D ; Depleted protein diet(rise powder 85% and no casein) 2) Group L ; Low protein diet(rice powder 80%, casein 5%) 3) Group M ; Moderate protein diet(rice powder 70%, casein 5%) 4) Group H ; High protein diet(rice powder 60%, casein 25%). Olive oil(4%). inorganic salt mixture(4%), cod liver oil(2%) and yeast(5%)were added in each diet with the same rate. (Salt mixture was made by the prescription of H.M. Wakeman, 1937) 4. Animal grouping of 90 rats were divided into 4 groups: 30 rats in group M and 20 rats each in groups D, L and H. Forty to 80gms diet was supplied to all rats every other day. The ingested and residual volumes of the diet were checked. The weight of the rats was measured from the day before the initiation of experimental feeding up to the date for the experiment to be finished. 5. Inoculation technique: E. histolytica was inoculated into the experimental rats which were fed by the experimental diets for 15 to 17 days. Amoebae were pooled from 48-hour-old culture tubes and spun for 5 minutes at 800-1,000 rpm. The sedimented amoebae were suspended in sterile saline containing 200,000 organismsper ml. The rats were anesthetized with ether, an incision was made slightly to theleft of midline of the abdomen, and the cecum partially exposed. Through a 5ml. syringe and a 23 gauge needle, the inoculum was injected toward the blind end of the cecum form a point anterior to the junction of the cecum and colon. The abdominal muscles and skin were then sutured with the surgical silk. 6. Paper electrophoresis of the rat serum protein: Rat sera were obtained from the cut tail using heparinized capilary tubes. For electrophoresis studies the sera were then placed in the Spinco electrophoresis chamber(Beckman Co.) on cellulose polyacetate strips in veronal buffer solution 9pH; 8.6) Each strip was stained in Ponceu S solution for 30 minutes. Thereafter serim protein fractionation was carried out by the elurion techique. The optical density of each fraction was calculated in the spectrophotometer at light length of 540mμ. 7. Identification of the pathogenicity of E. histolytica in experimental rats: The rats were sacrificed with ether 14 days after inoculation of E. histolytica. The entire cecum was removed and opened. The cecal content was examined for the presence of amoebae in a direct saline wet mount. The number and size of the crater-like ulcers in the ileoceal area were measured under the microscope, and then histo-pathological studies were carried on. The ulcers were divided into 3 degrees of severity of the ulceration as follows: Degree Ⅰ : One pin point ulcer in ileocecal area of rat. Degree Ⅱ : One to 2 ulcers of 1-2 mm in diameter Degree Ⅲ : More than 2 ulcers. Results and summary 1. The growth rates of rats according to the various diets. The growth of the depleted protein diet group(D) was markedly reduced more than that of any other group from 3rd day of diet control. The amount of ingested diet in the various groups did not show any difference statistically. An average amount consumed was 30-40gms day per capita. 2. Detection rate of E. histolytica trophozoites in each group. All the experimental rats were sacrificed 14th day after the inoculation with E.histolytica. Amoebae were found from the contents in the ileocecal area of the rats as follows: 100% in group D; 85.7% in group L; 736% in group M; and 44.4% in group H. 3. Electrophoretic fractionation of serum protein in rats. The serum protein fraction in each group before and after inoculation of E. histolytica is shown in the following table. Generally the γ-globulin level was increased in all the groups after the inoculation of amoebae, specially in hyperprotein diet group. 4. Total serum protein. The value of total serum protein in Group D (6.87gm%) was the lowest in any group. 5. Histological findings. ---------+---------------------------------+-----------------------------------0.752.7 Group H1.181.2 Group M0.910.952.5 Group L1.190.96---------+----------------+----------------+----------------------------------- Group DAfter inocula.Before inocula.----------------+----------------+The increase rates of γ-gl.(%) Groupsrestrictio