55. Inoculation Experiment o f Leprosy Bacilli with Liquid Paraffin and Subcutaneous Fat o f a Human Being or o f a Fowl.

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1 No. 8.j (8) Inoculation Experiment o f Leprosy Bacilli with Liquid Paraffin and Subcutaneous Fat o f a Human Being or o f a Fowl. By Saburo SATO. Kosankinbyo-Kenkyusho (Research Institute for Tuberculosis and Leprosy), Tohoku University, Sendai. (Comm. by T. KUMAGAI, M.J.A., Oct. 12, 1950.) Introduction. In an earlier paper I reported on marked leprous lesions produced in the visceras of domestic fowls which had been inoculated with emulsified leprosy nodules in a physiological saline solution into the pectoral muscles several times. The most marked and characteristic ones were formations of tumore-like leprous granulomas on the surface of the heart, liver, and kidneys, etc. In a cock, there was even the outbreak of two skin lepromas of plum size on both cheeks and a yellowish brown coloured macula of egg size on the neck were observed. However positive results occurred in only about 40% of these experiments, which means that we got only some 120 positive cases among about 300 fowls. Although successive subtransplantation tests of these lesions from fowl to fowl have not yet been fully successful, I was recently able to observe two cases, in which the same tumor-like lepromas were produced at the apex of the heart or on the liver, in the second generation of subtransplantation tests. In the meantime, I have got almost similar results with much more certainty in the inoculation tests, especially in fowls, which were infected with a suspension of leprosy bacilli (human and rat) in liquid paraffin or subcutaneous fat of a human body or of a fowl into the pectoral muscles. The results obtained will be reported in this paper. Method of Experiment. An aseptically excised leproma was cut with scissors in pieces as small as possible and ground in a glass morter ; then liquid paraffin or subcutaneous fat oil of a human being or a fowl was added to it (20-30 tunes in volume) and stirred up well ; the supernatant oily part was removed to a conical glass with a pipette and let stand for a while ; 2--4 c.c. of the supernatant oil was then injected deep into the muscle on each side of the breast of fowls.

2 (8) 46 S. SATO. [Vol. 26, Subcutaneous fat of a human being which we got by an autopsy and of a fowl, was melted by heating. After this stood for a while the oily part was removed and used. It is called " fat oil " in this paper. Dogs, rabbits, rats and mice were mostly inoculated in the testis ( c.c.). The second inoculation was carried out similarly ca 2-42 weeks after the first. Sometimes two inoculations of a suspension of bacilli in liquid paraffin (S.P.) were performed, sometimes once S.P. and then suspension in subcutaneous fat oil (S.O.) was injected. But in a case of human leprosy, human fat and in a case of rat leprosy, the fat of a fowl were used respectively. Besides those, S.P. or S.O. was injected either in fowls, which had been previously several times inoculated with suspension of bacilli in physiological saline solution (S.S. ), or in some cases S.S. was injected intravenously between two inoculations of S.P. and S.O. Results of Experiments. The results carried on so far are shown in the following tables. Among 13 fowls 6 died, one was killed and 6 are now yet alive. Among those that died or were killed 7 fowls had very marked or moderately marked lesions in 5 cases and in 2 cases only slight lesions. None of them was found without a lesion. By observing 6 fowls which are now living we find that in 3 cases positive skin reaction to lepromin of human or rat leprosy bacilli occurs. One killed fowl also has shown a positive reaction. These are far better results than those secured in the former experiments. Concerning macroscopical findings produced in fowls, as shown in Tab. 1 and 2, marked and characteristic lesions were seen in the viscera, especially in the heart and liver, of almost all the experimented fowls autopsied. In one case, No. 171, the lesions produced in the left lobe of the liver were very remarkable, showing numerous miliary or rice grain lepromas throughout the whole lobe. Any way, the characteristic lesions, which I found, are formations bf granulomatous neoplasmas on the surface of the viscera. They were brownish grey white or yellowish brown in colour and manifold in their shape and extent. They were often scattered on the epicard, the perihepar (peritoneal) membrane, or mesentery, as rice or bean grain sized nodules (No. 159, 167, II 160-4, II 160-2, 168).

3 No. 8.j Inoculation Experiment of Leprosy Bacilli with Liquid Paraffin etc. (8) 47 In the most characteristic cases, however, the greater part of the heart and liver were enveloped with thick greyish white granulomas of manifold shape and extent (No. 160-Fig. 1). Generally speaking, the granulomas showed an inclination for being produced in the thick capsule extending from the pericard to the interlobar and periportal part of the liver. Histologically the granulomas consist of many histiocytes, in which bacilli are often included, surrounded with numerous round cells. Among them small foci of necrosis and many vacuoles, which are due to fatty degeneration, are also marked. But sometimes Table 1. Experiment on human leprosy bacilli.

4 (8) 48 S. SATO. [val. 26, Table 2. Experiment on rat leprosy bacilli. the granulomas show a so-called tuberculoid state, consisting chiefly of infiltration of epitheiioid cells with central necrosis. Acid-fast bacilli were found, sometimes easily and numerous, even with lepraglobi (No. 167, 171) and sometimes scanty (No. 160, II 160-1) in the smears of the granulomas. But they were often found changing mostly their forms to granular ones. Meanwhile 2 hens had been inoculated with S.P. and one more hen inoculated with S.O. only one time, and autopsied 65, 92 and 18 days after the inoculation respectively. Almost no macroscopical changes occured in the viscera of all these cases. Next I have to tell about the skin allergy to leprosy bacilli (Mitsuda's or lepromin reaction), provoked in the experimented fowls. Naturally skin reaction to leprosy bacilli (human and rat) is always negative in untreated fowls. Moreover, I have always been assured that it remained quite negative in fowls, which were inoculated with S.S. repeatedly, even 10 times or oftener, into the pectoral muscles. However, it was found that skin allergy to leprosy bacilli can be provoked in some of the fowls, treated with

5 No. 8.1 Inoculation Experiment of Leprosy Bacilli with Liquid Paraffin etc. (8) 49 oil suspension, as shown in Tab. 1 and 2. But only one inoculation. alone was not enough to provoke this skin allergy in fowls even with oil suspension. The skin allergy seemed to be specific and there exists a distinct difference in fowls between an allergy to human leprosy bacilli and that to rat leprosy bacilli. Dogs, rabbits, rats and mice were inoculated with S.P. intratesticularly, once or repeatedly. Sometimes S.O. was injected into rats subcutaneously. But no marked lesions were observed in the viscera of any of the animals, except in the inoculated parts. Especially in rabbits, almost no marked lesions appeared in the viscera, as Saenz, Rist and Oka etc. have observed by inoculation of heat killed tubercle bacilli, although once or several times inoculations of S.P, were performed. Skin reaction to leprosy bacilli (human and rat) was examined in the case of many inoculated rats, but none of them showed a positive reaction. Discussion. Although the characteristic lesions in the viscera could be produced in fowls, inoculated with leprosy bacilli in a saline solution repeatedly, the results were surer and more marked, if the oil suspension of bacilli instead of the saline solution of bacilli was used. And moreover.two inoculations of S.P, or S.O., or S.P, and S.O. alternately, were enough to produce such lesions. Inoculation experiments of leprosy in fowls were formerly performed by Ota & Sato, Watanabe, Nonaka, and Ota & Nitto etc. They reported only the formation of granulomatous lesions in the injected muscle and miliary foci of infiltration in the parenchym of the liver and spleen etc. For the first time I was able to produce granulomatous neoplasmas on the surface of the heart and liver etc. besides the above known changes. However, for the production of these leprous neoplasmas, generally more than 5-6 times repeated injections of leprosy bacilli in saline solution were necessary. When we used a suspension of leprosy bacilli in an oily two injections were sufficient to provoke the leprous neoplasmas in the viscera and even a skin allergy to leprosy bacilli which was never observed formerly. Summary. 1) If suspensions of paraffin or subcutaneous inoculated at least twice leprosy bacilli (human and rat) in liquid fat oil of a human being or a fowl are into the pectoral muscles at certain inter-

6 (8) 50 S. SATO. [Vol. 26, vals, manifest tumor-like granulqmas were produced in the viscera, especially in the pericard, epicard, apex of the heart, pen- and interlobar capsule of the liver and mesenthry. Although similar lesions are produced by repeated injections of a suspension of bacilli in saline solution only, results obtained by this method were surer and more remarkable. 2) Skin allergy to lepromin (1:20 emulsion of leproma in saline solution) was provoked in some fowls, injected with an oil suspension of bacilli, while it remained always negative even in fowls, inoculated with a suspension of bacilli in a saline solution ten times and oftener repeatedly. Skin allergy to human leprosy bacilli is obtained only in fowls, inoculated with the same bacilli, and that to rat leprosy bacilli produced only in fowls and not in rats, treated with rat leprosy bacilli. And tuberculin skin allergy is not produced in all of the experimented fowls. (This study was aided by grants from the Ministry of Education. Acknowledgments are due to Prof. Dr. Kumagai, the former President of Tohoku University, for his advice and to Prof. Dr. M. Ito, Chief of the Dermatological Clinic of the University and Dr. Kamikawa, Chief of the Leprosari um, Tohoku Shinseien, for their aid). References. 1) Nonaka, N.: Kitasato Arch. Exp. Med., 1940, 12, ) Oka, S.: Kokenshi, 1946, 1, 25 (Jap.). 3) Oka, S. et al._: Sci. Rep. RITU, c, 1949, 1, 14. 4) Ota, M. & Nitto. S.: Jap. J. Exp. Med., 1940, 24, 1290 (Jap.). 5) Ota, M. & Sato. S.: Intern. J. Lep., 1940, 8, 81. 6) Rist, N.: Ann. Inst. Past., 1938, 61, ) Saenz, A.: C. r. Soc. Biol., 1939, 131, ) Saenz, A. et G. Canetti : Press. Med., 1939, ) Sato, S.: Sci. Rep. RITU, c, 1949, 1, ) Watanabe, Y.: Lepro, 1941, 12, 607 (Jap.).

7 No. 8.] Inoculation Experiment of Leprosy Paeilli with Liquid Paraffin etc. (8) 51 Fig. 1. Formation of granulomatous neoplasmas on the surface of the viscera in the fowl No. 160: a...of pericard; b..., of interlobar capsule; c, d... of perihapar capsule; e... of gizzard. Fig. 3. Histological view of the eosin, 10x40. liver in the fowl No. 171 Hematoxylin-