It has been found that although there is some parallelism between the quantity of tubercle bacilli demonstrable histologically and the number of colonies that can be isolated from a given tissue, the culture method is far the more efficient in indicating quantitative relations. Tubercle bacilli were not perceived in the organs of rabbits 1 day after infection with the modified BCG although as many as 1,500 colonies were isolated from one of them. This may be solely because it is difficult to see widely dispersed single minute acid-fast rods in the diffuse infiltrations of mononuclears with their hyperchromatic nuclei and sparse cytoplasm. Later, with the formation of tubercle, the parallelism is much closer. The culture method gives evidence concerning the number of living tubercle bacilli in the tissue.
The significance of the accumulation of acid-fast particles in the tissues has been discussed. It has been seen that from the beginning this accumulation is greater in the Kupffer cells of the liver, in the macrophages of the spleen and in the reticular cells of the bone marrow than within the mononuclears of the lung, the organ where the bacilli grow with the greatest rapidity and are destroyed with the greatest difficulty. Acid-fast particles are more prominent with the bovine than with the human bacillus or the BCG, the microorganism that is destroyed with the greatest difficulty thus leaving more incompletely digested bacillary debris at a given time within the cells. Thus it seems permissible to conclude from the presence of acid-fast material that some tubercle bacilli are undergoing destruction even 24 hours after infection. The initial accumulation of polynuclear leucocytes corresponds with the subsequent severity of the infection. Despite the greater primary localization of bacilli in the liver, this initial inflammatory reaction with all three infections is much greater in the lung than in the liver. In each organ it is more intense with the bovine than with the less virulent strains.
The multiplication of the bacillus and its accumulation within large mononuclear and young epithelioid cells is accompanied by an intense formation of new mononuclears by mitosis. The more rapid the growth of the bacillus, the more conspicuous the regeneration of these cells. Thus with all strains mitosis is more intense in the more susceptible organ, as in the lung compared with the liver; with the most virulent strain the most extensive and diffuse accumulation of these new cells corresponds with the greater rise in the numbers of bovine bacilli after the lag of the 1st week.
With the maturation of the epithelioid cells and the formation of tubercles the bacilli have already been greatly reduced numerically and the speed of this process diminishes with the virulence of the three strains used. The faster the development of tubercle the faster the destruction of the bacillus and the earlier the resorption of the tubercle.
Tubercle bacilli never accumulate in such large numbers in the mononuclears of the liver as they do in the lung. Though at first the tubercles in the liver may be more numerous than those in the lung they never attain the same size. The formation of new mononuclears by mitosis is restricted and Langhans' giant cells appear very early (1st and 2nd weeks). In the lung, giant cells are not found until much later with the BCG and the human bacillus (4th week); they were not noted in the interstitial tubercles with the bovine type, but the extension of these tubercles was accompanied by an unabated mitosis of mononuclears until the death of the animal. The liver tubercles are resorbed early even with the bovine infection. Associated with these histological differences are the slow initial growth and the early and complete destruction of the tubercle bacilli even of bovine type in the liver, and the more rapid initial growth in the lung, with the later destruction of the BCG and the human bacillus and the unabated growth of the bovine bacillus. Similar differences were observed between the splenic pulp and corpuscle. In the former the accumulation of acid-fast particles was much greater and the tubercles developed earlier. Mitosis of mononuclears was less frequent and giant cells appeared earlier. Tubercle bacilli, always intracellular, disappeared from the tubercles in the pulp sooner than from those in the corpuscle, and the tubercles themselves first disappeared from the pulp. Consequently with the persistence of bacilli mitosis continued in the tubercles of the corpuscle and these attained a much larger size.
Moreover individual resistance is linked with the ability to form mature tubercles early. In two animals simultaneously infected with the same strain and killed at the same time, the destruction or retardation of the bacillus is greater in that rabbit in which maturation of the tubercle and of epithelioid cells has proceeded further (Figs. 15 and 16).
These observations indicate that the mononuclears of different organs or even of the same organ, as in the different parts of the spleen, have a different capacity to destroy the tubercle bacillus, and that the transformation of the mononuclear into the mature epithelioid cell follows its destruction of the tubercle bacilli.
In the lung the more virulent types of bacillus are destroyed within the epithelioid cells of interstitial tubercles but persist in foci of tuberculous pneumonia. In this organ in rabbits infected with the human strain and to a lesser degree in rabbits infected with the bovine strain, the parasite largely disappears from the epithelioid cells of interstitial tubercles. But with both strains tubercle bacilli in large numbers may accumulate within epithelioid cells lying free in the alveoli. With the human type they are numerous within the cells and free in caseous material in the localized foci of caseous pneumonia. With the bovine infection, this caseous pneumonia is more often widespread and in the areas of caseous pneumonia the greater part of the vast accumulation of bovine bacilli in the lungs is found; as many as 200,000 colonies have been isolated from 10 mg. of tissue (Fig. 11). Flooding of the respiratory passages by the caseation of tuberculous lesions into the bronchi plays an important rôle in dissemination of tubercle bacilli through the lung. The process on the contrary is predominantly interstitial when the bovine bacillus is held in check (Fig. 12).
Thus there is apparently some factor acting in the alveoli that favors the growth of the parasite. The accumulation of tubercle bacilli is seen especially in the peripheral epithelioid cells in immediate contact with the alveolar space. In the same lung the bacilli are much fewer in the interstitial tubercles.
The accumulation in human tuberculosis of large numbers of tubercle bacilli in the tissues lining cavities is well known. Novy and Soule (20) have shown that within certain limits the growth of the bacillus in vitro is proportional to the oxygen tension of its environment. Corper, Lurie and Uyei (21) have confirmed these observations and have noted further that a difference in the gaseous environment of the bacilli equal to the difference between the conditions existing in the alveolar air and the venous blood is sufficient to cause a considerable increase in the growth of the microorganism in vitro. Loebel, Shorr and Richardson (22) by the use of Warburg's manometer have found that the oxygen consumption of tuberculous tissue is such that a tubercle 0.5 mm. thick would completely exhaust the oxygen of the air before it reached the center.
These observations suggest that a factor responsible for the greater multiplication of the bacillus in the cells of the alveoli may be the greater oxygen tension of the alveolar air.
In the liver, spleen and bone marrow even with the bovine infection many instances were found of the effective destruction of the parasite synchronously with the maturation of epithelioid cells and the formation of tubercle. On the other hand, in the spleen and bone marrow of some rabbits, living bacilli persisted within the epithelioid cells of isolated tubercles even 2 months after infection, a condition never found with the human type or BCG infection. Thus the epithelioid cell is the means of defense for the rabbit against the bovine type bacillus, and as such it is usually adequate in the liver, spleen and bone marrow though ineffective in the lung and kidney. In the latter, descending infection, and the occasional colony-like multiplication of bacilli in unorganized material, tubular casts, determine the long persistence of large numbers of bacilli in this organ.
In differentiating the mononuclear phagocyte of the connective tissues into the monocyte and clasmatocyte Sabin and her coworkers (23) have maintained that the clasmatocyte can efficiently destroy the tubercle bacillus but that the monocyte and its derivatives, the epithelioid and Langhans' giant cells, cannot. With the progress of the disease they have noted that the monocytes accumulate in great numbers in the foci of infection and overflow into general circulation (4). White (24) and Sabin and her coworkers have concluded that tuberculosis is specifically a disease of the monocyte, and that this cell and its derivatives act as incubators for the tubercle bacillus. Doan and Sabin (25) have therefore sought, with indecisive results, to protect the body against tuberculosis by an antimonocytic serum. However it has been shown here that although an intense multiplication of mononuclears is associated with the growth of the tubercle bacillus, their transformation into mature epithelioid cells is constantly associated with its destruction, and the rapidity of the destruction varies with the rapidity of the maturation of tubercle. Even in the bovine infection the epithelioid cells destroy the bacilli in the liver, spleen and bone marrow as a rule, and even in the lung, keep them in check in the interstitial tubercles.
The appearance of giant cells is associated with cessation or diminution of mononuclear regeneration by mitosis, and is coincident with cessation of multiplication or marked reduction in the number of living bacilli. They therefore appear earlier and in larger numbers in these organs or parts of organs that first destroy the bacillus (Figs. 16 and 17). They were not observed even 2 months after the bovine infection in the interstitial tubercles in the lung. Their absence and the continued mitosis of mononuclears, which accounts for the massive pneumonic and interstitial consolidation of the lung with this infection, were associated with the failure of the lung to destroy effectively the bovine parasite. The formation of giant cells in the pneumonic foci in the bovine infection would seem to be an exception to this rule.
The Langhans giant cells have often been considered an indication of the chronicity of the pathological process. It would appear that they are formed from existing epithelioid cells when the multiplication of the bacillus has ceased and the stimulus for the formation of new cells has decreased or stopped. Giant cells were most conspicuous in the liver and splenic pulp where, with the BCG infection, no caseation ever developed, and in the liver before caseation was seen anywhere in the body. In the human and bovine infections, giant cells formed in the liver before caseation appeared. Hence caseation is not a necessary requirement for giant cell formation, as maintained by Medlar (26), though these cells frequently form about caseous material.
Lymphocytes and granulation tissue do not cause the destruction of tubercle bacilli, these being destroyed in their absence. They usually appear about tubercles due to all strains and in all organs, after the greater part of the microorganisms have been destroyed (Fig. 18). The bacilli are not destroyed in the lung with bovine infection where the tubercles are usually little permeated by lymphocytes and granulation tissue. There is however, no constant relation between granulation tissue and destruction of tubercle bacilli, for in the lung after the human infection and even in other organs after the bovine infection isolated tubercles may be surrounded and penetrated by lymphocytes and granulation tissue at a time when considerable numbers of living bacilli are still histologically demonstrable within the epithelioid cells.
Caseation is usually not caused by the local accumulation of tubercle bacilli. At first, when the BCG (after 1 week) and the human microorganism (after 2 weeks) are present in the cells in very large numbers as demonstrated both histologically and by culture (Figs. 4 and 13) there is no necrosis of these cells. An exception to this rule found in the lung with the bovine infection is considered below. Later, after the bacilli have been destroyed to a great extent and even though the number of bacilli is small, caseation appears (Fig. 14). After this preliminary destruction the extent of caseation apparently varies with the number of residual bacilli. With the least virulent microorganism, the BCG, few bacilli remained in the liver in the 4th week and no caseation was seen. In the tubercles of the splenic corpuscle at the same time bacilli were somewhat more numerous and there was scant caseation. On the other hand with the human bacillus after 4 weeks more bacilli survived and caseation was more extensive in both organs; with the bovine microorganism tubercle bacilli were much more numerous and caseation was far advanced.
In the lung, however, caseation appeared with the first considerable accumulation of the bovine bacilli present 2 weeks after inoculation. That the bovine bacillus is primarily more injurious to the lung of rabbits than the BCG or the human bacillus is suggested by the greater intensity of the initial inflammation and by the more conspicuous accumulation of cells in the alveoli evident from the very beginning of infection. Maximow (27) showed that bovine bacilli even in small numbers cause the death of cells in tissue cultures of rabbit lymph nodes whereas the BCG or the human bacillus may accumulate within the cells in tremendous numbers without injuring them. Nevertheless in the liver, spleen and bone marrow of the living animal, caseation does not appear at the time when bovine bacilli are most abundant, but after they have been greatly reduced in numbers.
Large numbers of the less virulent types of tubercle bacilli accumulated in different organs a short time after infection do not cause caseation, and with the bovine infection caseation under the same conditions occurs only in the lung. Later when the animal is sensitized caseation occurs in various organs in the presence of the small numbers of tubercle bacilli that remain in the tissues after most of them have been destroyed, and the extent of this caseation varies with the numbers of residual bacilli. These observations suggest that a large number of bacilli fail to cause necrosis soon after infection whereas a few bacilli produce caseation in the animal that is sensitized.
Many investigators have held that caseation is due to sensitization. Krause (28), Huebschman (29) and Pagel (30) think that caseation is caused by the action of tuberculin-like substances on the sensitized tissues of the allergic animal. Rich and McCordock (31) view the process in essentially the same light. Recently Schleussing (32) has suggested that caseation is a coagulation necrosis in Weigert's sense of an allergically inflamed tissue, and is similar to the necrosis of the Arthus phenomenon.