마우스의 서라균 감염에 대한 병인론적 연구

Abstract

[한글]

[영문]

Rat leprosy bacillus, i.e., Mycobacterium lepraemurium, discovered by Stefansky in 1903 has never been successfully grown on bacteriological media, and therefore, animal inoculation was the method of choice for cultivation of the organism. Recently, growth of M. lepraemurium has been observed in tissue culture systems (Chung and Neikirk, 1965; Garbutt, 1965; Chang et al., 1967; Yang and Lew, 1968; Yang et al., 1968b).

The applicability of M.lepraemurium as a study of human leprosy has been frequently advocated (Carpenter, 1951; Tanimura, 1953; Lew, 1955; Rees and Waters, 1963), and accordingly, the organism has been extensively used as a experimental model in leprosy research.

Among the experimental animals known to be susceptible to infection with M. lepraemurium, rats have been exclusively used for animal transmission and extensive studies have been made on the pathogenesis and pathology of murine leprosy infection in rats (Hadler and Mauri, 1950; Hanks and Backerman, 1950; Nakamura, 1951; Hirai, 1952; Tanimura and Nishimura, 1952; Tanimura, 1953).

A variety of routes has been developed for animal inoculation of M. lepraemurium, i.e., subcutaneous, intracutaneous, intratesticular, intramuscular, intraperitoneal, intravenous and etc. (Tanimura, 1953).

Numerous reports have been published on the stuides of murine leprosy infection of rats following intraperitoneal inoculation (Stefansky, 1903; Lowe, 1937; Sellards and. Pinkerton, 1938; Kuwavarra, 1947; Hadler and Mauri, 1950; Hanks and Backerman, 1950; Nakamura 1951; Hirai, 1952; Tanimura, 1953). However, as pointed out by Hanks and Backerman (1950) there were rather wide discrepancies in the results of various investigators.

In sharp contrast to murine leprosy infection of rats, only a limited number of studies have been made on the experimental infection of the mouse with M. lepraemurium (Hanks and Backerman, 1950; Tanimura and Nishimura, 1952; Tanimura, 1953). Furthermore, no systematic studies have been carried out on the pathogenesis of murine leprosy infection of mice following intraperitoneal inoculation. The rationales for both intraperitoneal inoculation of M. lepraemurium into mice and suitability of such infected mice for the study of pathogenesis of murine leprosy infection are strongly supported by the following recent observations, i.e., 1) macrophages are the major host cells for the facultative and obligate intracellular pathogenic bacteria such as M. leprae and M. lepraemurium, M. tuberculosis,

Brucella and Listeria(Ptak et al., 1970; Godal et al., 1971), 2) there exist approximately 6x10**6 macrophages inside mouse peritoneal cavity (Kelly and Dobson, 1971), 3) the turn-over rate of macrophages in mouse peritoneal cavity is estimated at about 0.1% per hour and the turn-over time at about 40 days (van Furth and Cohn, 1968), and 4) all kinds of granulomas are made up of macrophages derived from circulating monocytes originated from the bone marrow (Spector, 1971).

By the aid of tissue culture technique and well-standardized counting of acid-fast bacilli by pin head method of Hanks (1968), pathogenesis of murine leprosy infection of mice following intraperitoneal inoculation was investigated in relation to 1) dynamics of multiplication of M. lepraemurium in peritoneal macrophages, 2) nature of splenomegaly and multiplication of M. lepraemurium in spleen tissues, 3) participation of bone marrow and peripheral circulation in the progress of murine leprosy infection, and 4) involvement of major reticuloendothelial system and lymphoid organs.

Materials and Methods

A. Materials.

1. Mycobacterium lepraemurium.

M.lepraemurium (Hawaiian strain) was inoculated into rat testicles, and the infected rat testicles 5 to 7 months after inoculation were used as the source of M.lepraemurium. The excised testicles were ground with mortar and pestle and a 10%

suspension was made with PBS or NCTC 135. The supernatant following centrifugation at 1500 rpm for 10 minutes of the 10% suspension of infected rat testicles was used as the inoculum in the mouse.

2. Mice.

Randomly bred mice of CFW strain, both sexes, weighing 15 to 17 gm of body weight were used. About 60 mice were inoculated intraperitoneally with M. lepraemurium and the number of M.lepraemurium inoculated per mouse was fixed at 2.3x10**6 in 0.1 to 0.2ml of the inoculum.

3. Tissue culture growth medium and miscellaneous solutions.

a) Growth medium: macrophage culture medium of Yang and Lew (1971) was used for tissue cultures of peritoneal macrophage and spleen cells, and it consisted of 2x NCTC 135 (Difco, U.S.A.); 5 parts, fetal bovine serum (Flow Laboratories, Inc., U.S.A.); 4 parts and diluted (1:5) bovine embryo extract; 1 part, and only

penicillin (500 u/ml) was added.

b) Phosphate buffered saline (PBS: Dulbecco); pH 7.2. Only penicillin (500 u/ml) was added.

c) Trypsin solution: 0.25% trypsin (1:250. Difco, U.S.A.) solution was made in PBS and Seitz filtered.

d) Heparin solution: 0.1% solution of heparin sodium (NBC, U.S.A.) was made in PBS and Seitz filtered.

e) NCTC-heparin mixture: NCTC-135 and heparin solution were mixed 9:1 just before use.

B. Methods.

1. Tissue cultures.

Following intraperitoneal inoculation of M. lepraemurium into mice, 5 mice were sacrificed by ether anesthesia at 24 hours, 1 week, 2 weeks, 4 weeks and 6 weeks, 2,3,4 and 5 months respectively, and tissue cultures of peritoneal macrophages,

spleen cells and bone marrow cells were conducted.

a. Peritoneal macrophage culture: The method used for collection of peritoneal macrophage was basically identical to thaat of Yang and Lew (1968) except that 3 ml of NCTC-heparin mixture was used for the 1st washing and 2ml for the 2nd washing.

For each culture experiment all of the peritoneal washing from the 5 mice were pooled and inoculated into Leighton tubes (1 ml/tube).

The inoculated Leighton tubes were incubated at 34℃, 5% CO^^2 atmosphere for 2 to 3 hours to facilitate macrophage adsorption to glass surface, and the NCTC-heparin mixture was replaced with fresh growth medium. Two more additional changes of growth medium were made at 1 and 4 days after initiation of tissue culture.

b. Spleen cell cultures: one third of each spleen was used for pathological preparation and the rest (2/3) was used for tissue culture. Combination of methods for lymphoid cell culture (Hellstrom and Hellstrom, 1969) and above-mentioned peritoneal macrophage culture was used for spleen cell cultures.

c. Culture of bone marrow cells: Right hind limb was amputated prior to collection of peritoneal macrophage for tissue culture, and bone marrow cells were obtained by washing out the bone marrow cavity of tibia with a small amount of NCTC-heparin mixture. One ml of pooled bone marrow washing from 5 mice was

inoculated into a Leighton tube and incubated at 34℃, 5% CO^^2, for 2 to 3 hours before fixation and staining.

2. Enumeration of number of acid-fast bacillus per cultures.

With Leighton tube cultures of peritoneal macrophages and spleen cells, the dispersed cell suspension was obtained by treatment with trypsin solution followed by mechanical scraping with rubber policeman. Then, the cells were disintegrated by ultrasonic treatment (Ultrasonic probe, BP-2 model, Fisher, U.S.A.) at 20KC for 4 minutes. The counting of total number of acid-fast bacillus per the cell sonicate was made by pin head method of Hanks (1968).

3. Preparations of peripheral blood smear.

Heparinized venous blood was obtained from the median canthus of the eye by the Pinkerton and Webber's method (1964) and smears were made on clean slide glass.

4. Preparation of pathological specimens.

Spleen, liver, mesenterium and mesenteric lymph nodes, thymus and axillary and inguinal lymph nodes were fixed with 10% formalin solution, processed, and stained preparations were made for pathological observation.

5. Staining.

The cells of peritoneal macrophage cultures, spleen cell cultures, bone marrow cultures, and peripheral blood smears were fixed with zenker-formalin solution and stained for acidfast bacillus by the method of Yang et al. (1968a). Pathological

preparations of spleen, liver, mesentery and mesenteric lymph nodes, thymus and axillary and inguinal lymph nodes were stained with hematoxylin-eosin and acid-fast stain. Preparations of bone marrow culture and peripheral blood smears were also stained with Wright stain.

6. Determination of splenic index (S.I.).

Splenic indices were calculated by the method of Simonsen (1962) as indicated below:

S.I.=mean experimental relative spleen weight/mean control relative spleen weight (Relative spleen weight=spleen weight/body weight)

Conclusion

1. A total number of acid-fast bacillus per culture of peritoneal macrophages decreased logarithmically for 2 months following intraperitoneal inoculation of M. lepraemurium into mice, and then increased sharply after 3 months.

2. In spleen active multiplication of M. lepraemurium occurred 3 months after inoculation, but until then there was no indication of such multiplication of M. lepraemurium observed in this organ.

3. It appeared that development of granulomas in the spleen was directly related to the phenomenon of splenomegaly. The granulomas consisted of aggregated masses of typical macrophages in which active multiplication of M. lepraemurium did occur.

4. Development of splenomegaly preceded active multiplication of M. lepraemurium in the spleen, and cultures of spleen macrophages made 4 months after inoculation exhibited significant proliferative activity in the tissue culture.

5. Infection of bone marrow cells was established very early and the extent of bone marrow involvement progressed steadily during the course of infection for 5 months. Nevertheless, bacillemia, in the form of either free acid-fast bacillus in

peripheral blood smear or acid-fast bacillus harboring moncoytic cells, were observed 3 months after intraperitoneal inoculation.

6. Certain differences were noted in the occurrence of leprotic lesions, the rate of their progresses, and the extent of multiplication of M.lepraemurium in liver, mesentery and mesenteric lymph nodes, thymus and axillary and inguinal lymph nodes. However, in later stages of the infection extensive developments of characteristic granulomas together with active multiplication of M. lepraemurium within the granulomas became manifest in all of the organs examined.