When animals are inoculated with an antigen during foetal or early postnatal life, a phenomenon known as immunological tolerance takes place which makes it difficult for the animal to produce antibodies against the same antigen when challenged after maturity.
The attempt was made to infect mice with the leprosy bacillus after eliminating the resistance utilizing this phenomenon.
The experiment was carried out a total of 4 times starting in March 1962. The mice were of the ICF strain and a total of 605 animals were used.
Inoculant : Lepromata from 4 untreated and 10 relapsed leprosy cases were collected, stored under refrigeration for 1 day to 70 days, sliced and an emulsion prepared with YLH solution. A small quantity of india ink was added. The inoculum contained 3×10
6-3.6×10
7 organisms per milliliter. For the pre-treatment antigen, the live bacterial suspension was used since it was believed that a degenerative change may take place in the bacterial cell component with heating. It need not be emphasized that all the procedures were carried out under sterile conditions.
Experimental Method: The animals were divided into two groups A and B.
The first group was further divided into 3 subgroups, I, II and III, according to the number of pre-treatments given and the control group, was divided into 2 subgroups, IV and V. I consisted of newborn mice and 0.05ml of live leprosy bacillus suspension was injected intraperitoneally within 24 hours after birth. II was treated in the same way as I but an additional injection of 0.01ml of bacterial suspension was given 1 week after the first. III was given 2 additional injections of 0.01ml basides the first at intervals of one week.
One to 3 weeks after the pre-treatment, 0.1ml of bacterial suspension was injected subcutaneously in the back. The animals were sacrificed after 10 - 20 months and examined for bacterial proliferation.
In IV, live bacterial suspension was injected intraperitoneally and subcutaneously in the back at the same time in the newborn mouse while in V, the live bacillus was injected subcutaneously in the 3-week-old animal without pre-treatment.
A piece of subcutaneous connective tissue was excised from lesions which could be observed macroscopically while in the animals in which no change was apparent, a piece of tissue was removed from the site of injection marked by india ink. A spread tissue preparation was made with the connective tissue specimen. Smears of the lyrnphnodes and organs were also prepared. These were stained with Ziehl-Neelsen's stain and examined microscopically for bacteria. Material showing proliferation of acid-fast organism was inoculated onto culture media and into the next generation of mouse, including both tolerance treated and non-treated animals. Two types of culture media, neutral medium for alkalinetreated and then neutralized material and acid medium, for alkaline-treated material were used. Cultivation was carried out at 33°C and 37°C.
From the findings in the present study, it is clear that pre-treatment to give immunological tolerance had no effect on the proliferation of leprosy bacillus inoculated in the mouse. Acid-fast organism which could be considered a true leprosy bacillus furthermore could not be isolated from any of the leproma material from 14 cases of leprosy. It should be noted, however, that numerous murine leprosy-like acid-fast bacilli similar to those reported by Nishimura were isolated. In the previous study, murine leprosy-like acid-fast bacillus grew in 14 of 711 suckling mice (ca 2%) inoculated subcutaneously with the live leprosy bacillus while in the present study, proliferation of an acid-fast organism which could be passed to other mice was found in 32 of 576 newborn mice (5.6%) inoculated intraperitoneally.
The reason for this increase of about 3% is not clear and further studies must be carried.
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