1) The fate of tubercle bacilli in rats when they are administered intra-

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1 STUDIES ON THE REINFECTION IN EXPERIMENTAL TUBERCULOSIS OF RATS KOOMI KANAI AND KEN YANAGISAWA The National Institute of Health, Tokyo (Received: January 11th, 1955) INTRODUCTION In the previous paper1), the reinfection in the experimental tuberculosis of guinea pigs was studied in various aspects. In the present paper, the reinfection experiment conducted similarly in rats was reported. It has been well demonstrated that rats are congenitally resistant to the infection due to tubercle bacilli by the investigations of Ornstein et al2), Boquet et, Nð gre3), Goldenberg4), Kato5) and so on6)7). Basing upon the data reported by them that tubercle bacilli can multiply considerably in those resistant rats, Lurie8) speculated that the hostparasite relationship between rats and tubercle bacilli may be symbiotic one. More recently, Wessels9) pursued the development of the experimental tuberculosis in rats through both histological observation and quantitative bacterial cultivation of various tissue. And he explained the antituberculous resistance of rats from his findings that tuberculin allergy did not develop and Gaseous lesions were not formed in those animals. On the other hand, however, the investigations to have shown the antimycobacterial immunity of rats in the reinfection experiment have not yet been reported hitherto, possibly due to the technical difficulties to differentiate the bacilli of primary from secondary infections. In this connection, experiments were undertaken in an attempt to determine whether or not an anti-mycobacterial power can be shown in tuberculous rats against secondly injected tubercle bacilli. The basic principle of the experimental procedure was just the same as that of the previous paper1). MATERIALS AND METHODS 1) Microorganisms: Three strains of tubercle bacilli, H37Rv, H37RvR-SM and 386R-INAH were used. The first two were sent from Dr. Karlson of the Mayo Foundation. The last was a pure isoniazid-resistant substrain obtained from the parent strain 386 which had been isolated in this laboratory from the sputum of a tuberculous patient. 2) The preparation of bacillary suspension, the method to evaluate the viability of those suspensions and the quantitative enumeration of viable organisms living in various tissue were described in detail in the previous paper. 3) Animals: Normal albino rats weighing approximately 100 g were used. EXPERIMENTAL 1) The fate of tubercle bacilli in rats when they are administered intra-, 129

2 130 KANAI et YANAGISAWA Vol. 8 venously in a large or small amount: The multiply and survival of tubercle bacilli in animal body are principally determined by the virulence of the bacilli and the intensity of the immunity produced in response to the bacillary multiplieating during the infectious process. Therefore, the analysis of the proliferation curve of the bacilli in rats may shed some light on the anti-mycobacterial immunity in these animals. With this point of view, an experiment was made as follows. Two groups of rats were used, each consisting of ten.the animals of one group were injected intravenously in the caudal vein with 0.1 mg of H37Rv (36 ~105 viable units) and those of the other group were treated in the same way with mg (36 ~103 v. u.) of H37Rv. The animals of each group were sacrificed two by two each one, fourteen, twenty-eight, forty-two and fifty-six days after injection, to conduct a quantitative enumeration of viable bacilli surviving in the spleen, liver and lung. The data were summarized in Fig. 1. Fig. 1. The fate of intraveneously-injected tubercle bacilli in rats 2) Comparative study of the fate of primary and secondly introduced tubercle bacilli in rates: It is a fundamental principle of tuberculous immunity that once infected individuals show a definite resistance against a subsequent infection. In this connection, an experiment was undertaken as follows in order

3 1955 EXPERIMENTAL REINFECTION TURERCULOSIS 131 to examine this kind of immunity in rats. Two groups of rats were arranged each consisting of ten. The animals of one group were infected with 25 ~103, viable organisms of the strain 386R-INAH. Three weeks after, all the animals of these two groups were infected with 4,500 viable cells of H37RvR-SM. Then, in just the same manner as before, the isolation of the primarily or secondly introduced tubercle bacilli was made at intervals of two weeks by sacrificing two animals of both groups each time. The differential isolation was possible using simultaneously Ogawa medium containing streptomycin or isoniazid in the concentration of 100 Ď/ml. The result was demonstrated in Fig. 2. Fig. 2. The fate of reintroduced tubercle bacilli in rats RESULTS AND DISCUSSION In Fig. 1, it is learned that the behaviour of tubercle bacilli to multiply in rats was different between in the lung and in the spleen or in the liver. During the observation period of eight weeks, the bacilli continued to multiply in the lung, but they ceased to increase in number in the spleen and liver in two or four weeks showing the almost horizontal growth curve thereafter. This tendency was the same as that reported by Wessels9). Thinking from the standpoint described just previously, the anti-mycobacterial immunity is considered to be manifested more remarkably in the spleen and liver than in the lung. This

4 132 KANAI et YANAGISAWA Vol. 8 is quite in contrast with the findings of Hashimoto10) in guinea pigs that the antibacterial immunity was most remarkably shown in the lung. In addition, macroscopical tubercles were found only in the lung, but they did not show any sign of caseation. The spleen was characterized with hyperaemia and swelling, but no tubercle was found. The spleen weight reached approximately 0.8 g in the animals inoculated with 0.1 mg of the bacilli. All the animals maintained the tendency of body weight to increase during the experimental period, but they did not react to a 1:10 dilution of OT in the examinations at any time. In Fig. 2, it is evidently shown that the reintroduced tubercle bacilli were markedly inhibited of their multiplication in the tuberculous rats, particularly in the earliar period of reinf ection. Of utmost interest was, however, the fact that this antibacterial immunity against reintroduced tubercle bacilli was manifested most intensely in the lung. Recently Dubos and his coworkers11) and Abe12) have also shown this type of immunity in the experimental tuberculosis of mice in which tuberculin allergy does not develop, claiming as an evidence for the view that the tuberculin allergy is one thing and the immunity is another thing. On the other hand, however, there has been no experimental evidence that the acquired immunity against tubercle bacilli develops in mice and rats in the same mechanisms as that in guinea pigs. Both tuberculin allergy and immunity develop on the basis of tuberculous infection. Therefore, if the host-parasite relationship between tubercle bacilli and the host differs, then the nature of the immunity may be different. Particularly in tuberculosis, the infectious process has been said to develop on the relationship between mononuclear phagocytes and tubercle bacilli. Recent investigations made by Brieger13), Suter,14), Mackaness15) have attached further significance to this view. In this country, Ogawa16) reported recently from his histopathological study that in guinea pigs the number of the monocytes which appear in response to the introduction of tubercle bacilli is large but the bacilli ingested by those cells are not so numerous. Contrary to this, the appearance of the monocytes is not so large in rats as in guinea pigs but the bacilli ingested are numerous. As well known comparing with our previous paper17), the rats maintained the healthy condition against the vigorous proliferation of tubercle bacilli in their bodies to such an extent as it killed the host in the case of guinea pigs. Wessels9) considered that the resistance of rats to tuberculous infection may be due to the fact that the cells ingesting tubercle bacilli do not come to necrosis and caseation. In this connection, the report18) that tuberculous rats take fatal outcome when they are treated with cortisone is of particular interest. Lastly, the author should like to think about the nature of the immunity in rats. The term gimmunity husually suggests a kind of biological phenomenon in which the antigen-antibody reaction is taking a part in a direct or indirect way. On the other hand, the phenomenon that bacterial cells multiply and then decrease in the number of viable cells occurs not only animal body but also in culture

5 1955 EXPERIMENTAL REINFECTION TUBERCULOSIS 133 medium. And in the latter case, the change of ph and the accumulation of metabolites in the medium may be principal factors responsible for the phenomenon. If we consider the interior of mononuclear phagocytes as culture medium, the fate of the bacilli ingested by them may be understood without paying any particular considerations to the antigen-antibody reaction in vivo. Recently, Dubos19)20) pointed out it important to think the infectious process from the physicochemical standpoint. At present, we should like to present a way of explanation for rat tuberculosis that the host-parasite relationship between rats and tubercle bacilli should be called gmutually indifferent h, even though it is not symbiotic, and the immunity-like phenomenon against tuberculous infection may be of non-antibody nature. SUMMARY 1) An antituberculous immunity was demonstrated in rats. 2) The nature of this immune power was discussed apart from the tradi tional antigen-antibody concept. REFERENCES 1) Kanai, K. and Yanagisawa, K.: Studies on the reinfection in experimental tuberculosis of guineaa pigs. Jap. J. Med. Sci. & Biol., 8, , ) Ornstein, G. G. and Steinback, M. M.: The resistance of the albino rat to infec tion with tubercle bacilli. Am. Rev. Tuberc., 12, 77-86, ) Boquet, A. et Nð gre, L.: Contribution a l'etude de l'infection tuberculeuse chez les petits rongeurs. Ann. Inst. Past., 35, , ) Goldenberg, I. J.: Beitrð ge zur Frage ð¹ber den Mechanisms der Immunitð t von Ratten gegen Tuberkulose. Z. f. Tub., 55, , ) Kato, G.: Further studies on the experimental tuberculous infection. I. Ex periments in rats. Kekkaku (tuberculosis), 20, 71-84, 1941 (in Japanese). 6) Hussel, L.: ð ber die Eignung des Goldenhamsters als Versuchstier bei der Tuberkulose-Diagnostik. Zentralbl Bakt., 1, 156, , ) Hagedorn, K.: Versuche an Ratten ð¹ber den Einfluss des Vitaminmangels auf den Verlauf der Tuberkulose. Beit. z. Klin. d. Tub., 70, , ) Lurie, M. B.: Native and acquired resistance to tuberculosis. Am. J. Med., 9, , ) Wessels, C. C.: Tuberculosis in rat. I & II. Am. Rev. Tuberc., 43, , ) Hashimoto, T.: Medicine and Biology, in press (in Japanese). 11) Dubos, R. J. et al.: Antituberculous immunity induced in mice by vaccination with living cultures of attenuated tubercle bacilli. J. Exp. Med., 97, , ) Abe, H.: Immunity experiments in experimental tuberculosis of mice. I. Effects of the vaccination with BCG or heat-killed tubercle bacilli. Kekkaku (tuber culosis), 28, , ) Brieger, E. M.: New approches to the study of experimental infection. Advances in Tuberculosis Research, 4, , 1951.

6 134 KANAI et YANAGISAWA Vol. 8 14) Suter, E.: Multiplication of tubercle bacilli within mononuclear phagocytes in tissue culture derived from normal animals and animals vaccinated with BCG. J. Exp. Med., 97, , ) Mackaness, G. B. et al.: The growth of intracellular tubercle bacilli in relation to their virulence. Am. Rev. Tuberc., 69, , ) Ogawa, H.: Histological studies on the susceptibility of host body to tubercle bacilli. (II) Nippon Byorigaku Zasshi, 42, 1-2, 1953 (in Japanese). 17) Kanai, K. and Yanagisawa, K.: Studies on the infection and immunity in ex perimental tuberculosis of guinea pigs. II. Relation between the virulence and the immunogenicity of tubercle bacilli. Jap. J. Bact., in press (in Japanese). 18) Roche, P. Jr. et al.: Comparison of experimental tuberculosis in cortisone treated and alloxan diabetic albino rats. Am. Rev. Tuberc., 65, , ) Dubos, R. J.: Effect of the composition of the gaseous and acqueous environ ments on the survival of tubercle bacilli in vitro. J. Exp. Med., 97, , ) Dubos, R. J.: Effect of keton bodies and other metabolites on the survival and multiplication of staphylococci and tubercle bacilli. J. Exp. Med., 98, , 1953.