Ogawa's Bacillus: Slow Growing Mycobacteria Isolated from Mice Previously Infected with Murine Leprosy Bacillus

I. In Vitro Cultivation and Animal Inoculation

Abstract

In 1969, Ogawa and Motomura¹⁾ isolated slow growing acid-fast bacilli from mice previously inoculated with the Hawaiian strain of Mycobacterium lepraemurium. The 4th subcultures of the organisms, designated here as strain HI of Ogawa's bacillus, were given through the courtesy of Dr. Ogawa and submitted to the present experiment in which comparative studies with other known strains of slow growing mycobacteria were carried out.
Growth characteristics in vitro: When heavily transferred by loop (Table 1), the organisms grew very slowly on Ogawa's 1% egg yolk medium¹⁾ but not on the 1% egg medium⁴⁾, the egg yolk medium deprived of glycerol, or the Morrison⁶⁾ and Smith⁷⁾ agar media containing mycobactin. The addition of heat-killed M. phlei cells to the egg yolk medium did not enhance the growth of the organisms. Small inocula from the filtered bacterial suspension, 0.01mg to 0.3mg (moist weight), yielded isolated colonies on the egg yolk medium; growth became barely visible to the naked eye after approximately 40 weeks' incubation at 37° (Table 2).
Animal test: Male mice of dd-Y strain, weighing 20 to 22g, were inoculated intravenously with an amount of 0.2 or 0.02mg of the 4th subculture and utopsied 5 to 10 months later. As shown in Table 3, the organisms gave rise to a progressive murine leprosy-like disease with severe lesions involving liver, spleen, and skin; the lungs were less severely affected. Most of the smears from these tissues revealed vast number of acid-fast bacilli. For isolation weighed tissue was ground in a mortar and treated with 5 to 10 volumes of 1 per cent sodium hydroxide; 0.1-ml amounts (20 to 10mg of tissue) of the resulting suspension were pipetted into Ogawa's egg yolk and egg slants. Of a total of 59 specimens inoculated, 35 gave positive cultures on the egg yolk medium, 19 negative and 5 contaminated. No positive cultures were obtained with the egg medium. All of the cultures recovered were identified with the organisms employed for injection.
It seems reasonable to conclude that strain HI is very characteristic in vitro and in experimental animals, and definitely distinguishable from other known culturable mycobacteria. Some similarities between Ogawa's bacillus and murine leprosy bacillus has briefly discussed.