Antigenic studies of mycobacterium leprae and mycobacterium lepraemurium by immunodiffusion
[영문]Since G.A. Hansen discovered M. leprae as a causative organism of human leprosy, many investigators have continued their efforts to cultivate M. leprae in vitro.
Although Shepard(1960) succeeded in multiplying it by animal inoculation in mouse foot pad, M. leprae still can not be cultured in artifical media. Therefore, immunologic studies of leprosy lag far behind those on other mycobacterial infections, and the identification and establishment of relationships among various antigens associated with mycobacteria have been the subjects of numerous investigations(Parlett and Youmans, 1956; Burrell and Rheins, 1957; Pepys, 1960; Tuma and Silva, 1962; Rees et al., 1965; Estrada-Parra et al., 1966; Abe, 1970; Navalka, 1970).
Recently the immunodiffusion methods have been widely used for the antigenic analysis of mycobacteria. Since Parlett and Youmans(1956) applied the agar gel diffusion technique to identify the antigenic relationship between mycobacteria, many investigators have developed and applied the immunodiffusion methods to antigenic analysis of M. leprae and other mycobacteria.
Burrell and Rheins(1957) studied antigenicity of lepromin and reported that the old tuberculin and lepromin were shown to possess common as well as distinct antigena. Pepys et al.(1959) and Pepys(1960) observed that PPD preparation and lepromin had three common precipitating antigens. Sushida and Hirano(1961a, b), Navalkar et al.(1964, 1965), and Navalkar(1970) detected antibodies in the sera from leprosy patients against culture filtrates of various mycobacteria and lepromin. Tuma and Silva(1962) obtained positive precipitating reaction between
various mycobacterial antigens and rabbit serum immunized with leprosy bacilli.
However, only a few papers have been reported on the specific antigens of M. leprae because of difficulties in obtaining enough amount of purified leprosy bacilli. Some investigators(Olmos Castro and Arcuri, 1958; Rees et al., 1965; Estrada-Parra et al., 1966; Abe, 1970) suggested that leprosy nodule contained certain specific antigens by the studies of lepromin, unheated leprosy nodules and tissue culture filtrates of M. lepraemurium. Rees et al.(1965) reported that tissue culture filtrates of M. lepraemurium contained soluble antigens comparable to those
in the culture filtrates of other cultivable mycobacteria and they assumed that the intracellularly growing M. leprae and M. lepraemurium in vivo were equally able to produce soluble antigens which could be released into the surrounding tissue space or into the blood stream. Abe(1970) isolated a water soluble, heat labile, protein antigen from unheated leprosy nodules, which gave precipitation only with the rabbit anti-leprosy nodule-extract serum. Those studies suggested that leprosy nodule and murine leprosy infected tissues contained certain specific antigens.
The present paper reports the antigenic analysis of unheated human leprosy nodule extract, purified leprosy bacilli, murine leprosy infected rat testicle extract and purified murine leprosy bacilli, and the detection of antibodies in the sera from leprosy patients various mycobacterial antigens.
Preparation of Antigens:
Human leprosy nodules were obtained from lepromatous leprosy patients who were under antileprosy treatment at World Vision Special Skin Clinic in Seoul, and normal human skin was collected from cadaver at Severance Hospital. Murine leprosy infected rat testicles were obtained from rats infected with murine leprosy bacilli by inoculation into testicles nine months before.
Tissue extracts from human leprosy nodules, normal human skin, murine leprosy infected rat testicles, and normal rat testicles were prepared as follows: each tissue was minced with scissors and homogenized with about 10 volumes of 0.2M sucrose solution in a glass tissue grinder for about 10 minutes. These suspensions
were centrifuged at 1,000rpm for 10 minutes and the supernatanats were further centrifuged at 10,000rpm for 2 hours at 4℃. The supernatants were dialyzed against distilled water for 48 hours at 4℃ and then concentrated to about one half of original volume with carbowax 6000(polyethylene glycol).
M. leprae and M. lepraemurium were collected, respectively, from the human leprosy nodules and murine leprosy infected rat testicles as follows: the homogenized suspensions of human leprosy nodules and murine leprosy infected rat testicles with 0.2M sucrose solution were centrifuged at 1,000rpm for 10 minutes
and the supernatants were further centrifuged at 10,000rpm for 2 hours at 4℃. The sediments were resuspended with phosphate buffer saline solution(pH7.2) and treated by a ultrasonic probe(fisher, Model BP-2) with 20 KC for 10 minutes. The residual
tissue debris in the bacillary suspensions were eliminated by digestion with 2 percent trypsin solution(1:250, Difco) in phosphate buffer saline for 3 hours at 37℃ and subsequently by repeated washings with distilled water. Finally, these bacillary suspensions ere concentrated to contain approximately 100mg wet weight of bacilli per milliliter, and disintegrated by a ultrasonic probe with 20KC for a total of 3 hours.
Culture filtrates of other cultivable mycobacteria, i.e., M. avium, M. bovis(BCG), M. kansasii, M. tuberculosis(H^^37 Rv), M. smegmatis, and M. phlei were prepared as follows: each strain of mycobacteria was cultured in Sauton media for eight weeks at 37℃, and the culture filtrate was obtained after filtration, first with filter paper and second with a membrane filter(S & S, 0.45μ). No heating was applied in these procedures. The culture filtrate was then dialyzed against distilled water for 48 hours at 4℃ and concentrated to approximately one-tenth of original volume with carbowax 6000.
Preparation of Antisera:
All antisera were obtained from white rabbits immunized with the prepared antigens.
Antisera to the human leprosy nodule and murine leprosy infected rat testicle extracts were produced by injecting the rabbits intramuscularly in each buttock and hind foot pad with a total of 2ml of prepared antigens suspended in Freund's incomplete adjuvant. After 4 times of incoculation at one week intervals, the immunized rabbits were bled an the immune sera were separated.
Antisera to M. leprae and M. lepraemurium were, respectively, prepared by injecting the rabbits intramuscularly in each buttock and hind food pad with the prepared mycobacterial antigens suspended in Freund's incomplete adjuvant. After 5 times of injection at ten days intervals, the immune sera were obtained.
Double diffusion -in-agar-gel was performed according to modified Ouchterlony(1958) method, using 100mm Petri dishes covered with 0.85 percent purified agar(Difco) in phosphate buffer saline(pH 7.2). Well patterns were varied to suit the requirements; the diameter of a well was 7mm and the distance between wells was 8mm. The age plates were incubated at 37℃ and the results were observed every day for four to five days.
Immunoelectrophoresis was carried out by the method described by Campbell et al.(1963), using 0.85 percent Agarose agar(Mann Research Laboratories).
1. The human leprosy nodule extract contained at least two kinds of antigens, one of which was rather specific, heat labile, and precipitated only against rabbit antileprosy nodule-extract serum and antileprosy-bacilli serum, but not contained in normal human skin extract and human serum. The other antigen reacted with the rabbit antileprosy-bacilli serum and shared commonly with M. lepraemurium and other mycobacteria.
2. M. leprae contained one or two common antigens with other mycobacteria, and two common antigens most closely related to those of M. lepraemurium.
3. The murine leprosy infected rat testicle extract contained at least three kinds of antigens, two of which were rather specific, heat labile, but not contained in either normal rat testicles or normal rat serum. The another one was a common antigen shared with other mycobacteria.
4. M. lepraemurium contained more than one or two common antigens shared with other mycobacteria, and two antigens most closely related to those of M. leprae and M. avium.
5. Two kinds of antibodies were detected in the sera of leprosy patients against various mycobacterial antigens, and among the various antigens tested M. lepraemurium was found to be particularly suitable for the serological investigations of leprosy patients.