Early Evaluation of the Multiplication of Murine Leprosy Bacilli Using Spread Tissure Preparation

Abstract

In the experimental murine leprosy the infection is usually produced by subcutaneous or intraperitoneal inoculation. Under these conditions the long period of observation, up to 2-3 months, is generally required. On the other hand, the observation period can be shortened by using bacillus counting as the criterion for evaluating the bacillary multiplication. However this method is very complicated technically. Therefore, it is desirable to find a simple and reliable method by which the experimental period should be shortened.
Since the good results were obtained on our works by using the spread tissue preparations to find acid fast bacilli in the subcutaneous tissues of healthy mice, this technique has been used in the early stage of subcutaneous infection to investigate the development of murine leprosy.
In this experiment, mice of the C57BL/6strain, being of benign type, were inoculated subcutaneously in their back with 0.25ml of a 1:1000 leproma suspension and sacrificed on 1, 5, 10, 15, 20 and 25 days to examine the spread preparations from the site of inoculation. One day after the inoculation, short acid-fast bacilli similar to the initial ones were found in cytoplasm of mononuclears. After 5 days, elongation of the bacilli was observed in mononuclears without increase in number. The maximum length of bacilli was about 2-3 times the initial size. On 10th day, fairly numerous bacilli of long forms were observed. This finding suggests that the bacillary multiplication has begun to occur. Marked increase in number of the bacilli was observed for 15 to 25 days after the inoculation. Therefore the enlargement of mononuclears, each of which was being crowded with longer bacilli, could easily be observed microscopically in a low magnification.
Elongation of the bacilli and increasing in number were examined in C3H mice, being of malignant type, in the same way. A pattern similar to those observed in C57BL/6 mice was also obtained. No significant difference was found between the mouse strains.
On the basis of these observations, this experimental method can be recommended for an early evaluation of development of murine leprosy.