한국산 다람쥐 (Tamias sibiricus asiaticus, Gmelin)를 사용한 인라균 증식실험
Experimental studies on the growth of M. leprae in the Korean chipmunks (tamias sibiricus asiaticus, gmelin)
Shepard (1960a, 1960b) observed that inoculation of M. leprae derived from nasal excretions and nodules of leprosy patient into foot pads of mice resulted in a consistent but limited growth of the organism. In the fellowing years Shepard's observations have been amply confirmed and extended (Shepard, 1962, 1963, 1965 a;
Janssens and Pattyn, 1963; Kirchheimer, 1964; Rees, 1964, 1965; Shepard and McRae, 1965; Rees and Weddell, 1970; Yang and Lew, 1971). The resolution that M. leprae would grow in mouse foot pad was officially made at the Ⅷth International Congress of Leprology, Rio de Janeiro in 1963.
It is now well accepted that M. leprae will grow to a limited extent in the mouse foot pad, and that Shepard's technic of foot pad inoculation has become a standard method of animal inoculation of M. leprae in the laboratory. In addition to the
mouse system, a similar pattern of growth of M. leprae has been observed in a number of other rodents (Niven and Waters, 1963; Waters, 1965; Waters and Niven, 1965; Hilson, 1965).
The mouse foot pad technic has been widely applied in the studies of leprosy, i.e., 1) screening and selection of antileprosy drugs and determination of effective dosages of drugs (Shepard and Chang, 1962; Shepard et al., 1966; Rees, 1967a; Shepard, 1969; Rees et al., 1970; Ozawa et al., 1971), 2) measurement of generation time of M. leprae (Shepard and McRae, 1965), 3) confirmation of the viability of M. leprae in regards of staining characteristics ("solid-staining") (Shepard and McRae, 1965 ; Rees, 1965), 4) identification of dapsone-resistant
strains of M. leprae (Rees, 1967b; Rees and Weddell, 1970) and 5) the evaluation of effectiveness of BCG vaccination as a prophylactic measure against leprosy infection (Shepard, 1965b; Shepard and Ribi,1968).
Though some features of generalized infection of mice with M. leprae have been observed in the thymectomized and total body irradiated mice (Rees and Weddell, 1970), a definite ceiling effect became evident on the maximal number of M. leprae grown in the font pads of immunologically competent mice (Shepard, 1960a ; Rees and Weddell, 1977).
Since 1967 we have been working on the experimental infection of the Korean chipmunks (Tamias sibiricus asiaticus, Gmelin) with M. leprae as a search for a better animal host in the study of human leprosy. Application of the Korean chipmunks as experimental animals for the growth of M. leprae is based on the
facts, i.e., 1) the chipmunks are readily available in large numbers and they can be easily maintained in the laboratory for experimental purpose, 2) an average life span of the chipmunks is known to be approximately 7 years which is far longer than that of mouse and 3) the chipmunks proved to be highly susceptible to experimental infection with M. tuberculosis (Chang et al., 1971).
In this communication we present the evidences of active growth of M. leprae in foot pads and ear lobes of the chipmunks, major characteristics of pathological changes in these inoculated tissues, and the result of clinical study in a series of leprosy patients of the chipmunk lepromin antigen prepared from infected
chipmunks' font pads.
Materials and Methods
Animals. Korean chipmunks of both sex, age of less than one year and 50 to 70 g of body weight were used throughout the experiments. At the laboratory they were maintained in metal animal cage for rabbit (8 to 10 chipmunks per cage) and fed
with boiled corn, chestnut, acorn, boiled pupa of silkworm, vegetable and water.
Prior to inoculation with M. leprae, the chipmunks were allowed to be accustomed to the laboratory maintenance for 2 to 4 weeks. Mice of CFW strain, both sex and weighing 18±2g were also used.
The temperature of animal room wart kept at 20±1℃.
M. leprae. Biopsied nodules from fresh untreated lepromatous leprosy cases were the source of M. leprae for animal inoculation. The nodules were used either immediately after biopsy or kept frezen at-15℃ up to 4 weeks before use.
Mincing with scissor (or scalpels)
Trypsinization*** for 1 hour at 37℃ with agitation by magnetic stirrer
Filter with 3 layers Of sterile gauze
Centrifuge at 1,000 rpm for 10 minutes
Centrifuge at 10,000 rpm for 1 hour at 4℃
Suspend in PBS****
Fig. 1. Trypsin purification of M. leprae from biopsied lepromatous nodules.
(*)**Trypsin solution: 0.25% in PBS
(**)**PBS (phosphate buffered saline, Dulbecco)
Two preparations of M. leprae were used for animal inoculation, the first one was prepared by conventional grinding method followed by light centrifugation, and the second by trypsin purification method as shown in Fig. 1.
Counting of numbers of acid-fast bacilli (AFB). The pin head method of Hanks (1968a)
Lepromin antigens. The standard lepromin was prepared from biopsied lepromatous nodules and the chipmunk lepromin from infected foot pads of the chipmunks by the method recommended by Hanks (1968b), and each of antigen preparations contained 160X10**6 AFB per ml.
Pathological preparation. The infected tissues were fixed with buffered formalin, paraffin embedded, sectioned and hematoxyline-eosin (H-E) and acid-fast (A-F) stained.
1. M. leprae, obtained from lepromatous nodules either by conventional grinding or trypsin purification method, multiplied in both foot pad and ear lobe of the Korean chipmunks through the first and the second passage experiments. Growth of M.
leprae in these inoculated tissues became evident after a lag phase of approximately 7 months postinoculation.
2. Characteristic leprotic changes were observed in foot pads of the chipmunks inoculated with trypsin-purified M. leprae 13 and 16 months ago, and these changes included extensive leproma formation, presence of massive numbers of acid-fast bacilli in the leproma cells and involvement of dermal nerve fibers by acid-fast bacilli.
3. Among the chipmunks inoculated with trypsin-purified M. leprae for the preparation of the chipmunk lepromin antigen, apparent swelling of the inoculated tissues was observed in a considerable number of the chipmunks at 10 months after inoculation. Two of such swollen foot pads contained 2.OX10**10 acid-fast bacilli respectively.
4. The results of skin tests in a series of leprosy patients with the chipmunk lepromin, prepared with acid-fast bacilli harvested from the swollen infected foot pads, were identical with those of the standard lepromin prepared from biopsies