THE EARLY REACTION IN THE INTESTINAL MUCOSA OF THE GUINEAPIG ' T0 INGESTED MYCOBACTERIUM ‘ TUBERCULOSIS OF THE BOVINE AND HUMAN TYPES THESIS FOR THE DEGREE OF M. S. Virginia Dufresne. Lauzun 1 1933 W4€z mam IFDK ~.Dl.‘ THE EARLY REACTION IN THE INTESTINAL MUCOSA OF THE GUINEA PIG TO INGESTED MYCOBACTERIUM TUBERCULOSIS OF THE BOVINE AND HUMAN TYPES. THE EARLY REACTION IN THE INTESTINLL MUCOSA OF THE GUINEA PIG TO INGESTED MYCOBACTERIUM TUBERCULOSIS OF THE BOVINE AND HUMAN TYPES. THESIS Submitted to the faculty of tha Michigan State College in partial fulfillnnnt of the requirements for to degree at Mastor d Scimo 1n.LnImal.Patholqu by Virginia Dun-a one Lanznn 1953 r “-45 1- H bi CONTENTS Introduction Review of Literature Experimental Gmup I Group II Group III Blood Examinaticne Cone illusions Appendix Bibliography Acknowledgment 100203 INTRODUCTION This work was undertaken to determine, if possible, if phagocytosis of tubercle bacilli occurs within the lumen of the intestine (as has been demonstrated for Johne's bacillus by Hallman and Witter) and to determine whether the phagocytes than carry the bacilli into the strama, or, if not, the manner in which the bacilli do enter. REVIEW OF LITERATURE In their work, "Some observations on the pathology of Johne's disease," (unpublished) E. T. Hellman and J. F. Witter (2) illustrate phagocytosed Johne's bacilli within the gland tubule of the cow's intestine.* They believe that this is indicative of intratubular phagocytosis of rods and not or exudation oi' rod-containing mcrophages into the lumen, because the incidence of its appearance is not common and, moreover, it appears no more frequently with "most extens ive hyperplasia with innumerable macrOphages containing masses of reds” than in very mild cases. Since the macrophage is capable of passing into the lumen, there seems no obvious reason why it could not retun in the same manner to the stroma, carrying its burden of acid- fast bacilli. The possibility of an attraction of the Johne's bacillus in the lumen for macrophages and for polymorphonuclear phagn. cytes (with which they are found associated, but within which the organism has never been seen by Hellman an! Witter) is sugested. From their work the authors believe that there is a probability that the primary lesion or Johne's disease may be intratubular, i.e., originating within the gland tubule. It therefore seemed desirable to determine whether the tubercle bacillus could be found phagocytosed in the lumen or gland tubules of the intestine althougi with the tubercle * In describing organi ans as being *phagocytosed" in this thesis, the term will be used in the same sense in which Hellman and Witter have used it - indicating ingestion, but not necessarily destruction, of the bacilli. bacillus this may occur with polymorphonuclear leucocytes as well. It was also desired to ascertain whether phagocytes containing the rods couhd be demonstrated within the epithe- lium, If this could be shown within.a few hours or’a.few days after ingestion of the bacilli, it would point to a pro- gress of the cell inward to the stroma.and not outward into the lumen, as might be conceived in later cases with obvious lesions in flhe intestinal well. Many papers have recently appeared on the early cellular reactions to injected bacilli'both in primarily and secondarily infected animals, but the actual entrance of the organisms, without tramna, has not been studied so extensively. Calmotte (3) states that tuberculosis never primarily invades the highly specialized tissue elements. The latter are involved secondarily through extension of seasons and sclerotic processes. He goes further to state that whenever a tubercle bacillus, deposited upon the skin surface or the mucosa or introduced into the healthy body in.any other manner, finds itself near a motile polymorphonuclear leucecyte, it inmo- distely becomes the pray at the latter, which takes it into the lymphatic or blood circulation. In the buceopharyngeal cavity, however, Calmette believed that any leucccytes thidh escaped from the tissues were prevmted from reentering the body because of the movements of swallowing. Here an excep- tion is made for the tonsils, where the crypts and spaces are "tightly packed with lymphocytes and polymorphonuclear leuco- cytes, and constitute by virtue of their position an.important defense mohanism. 'tubercle bacilli, however, when trapped in the crypts of the tonsils may use these as portals of entry. The primary lesion.may develop in the closed follicles of the tonsil and there progress to caseation and ulceration. The oesophagus, by reason of its stratified epithelium, and the stomch, because of the "dmsi ty" of the lining layer, and both by virtue of the secretions pouring out on their surfaces, Calnette states, are more resistant than the rest of the alimentary canal to tuberculous infection. On.the other hand, phenomena of absorption are accomplished with increasing intensity from the duodenum to the termination of the mall intestine and with diminishing intensity from the ilsccecal valve to the rectum. The intestinal epithelium permits wandering cells to ”penetrate between the cylindrical epithelial cells" and carry back into the dhyle-bearing passages bacterial bodies and certain mineral particles. This passage is most active at the level of Peyer's patches, abundant in the ileum, Fram.a variety of experiments reviewed by Calmette, it would seem that many bacteria, inclu- ding mycobacterium tuberculosis, pass through the mucosa more easily during the digestion of fatty substances. no experiments are cited demonstrating bacilli actually phagocytosed by polymorphonuclear or other cells in the process of passing through.the epithelium, On.the contrary, let us quote, "They (the tubercle bacilli) pass through the intestinal mucous membrane without leaving the slightest trace of their passage and when arrived at the chyle-bearing vessels of the villi are already found to be the prey for leucocytes." Nedseland Keller (4) recently published an article on the passage of exogenous bacteria throigh the wall of an isolated stomach. They cite Nedzel and Arnold as previously showing passage from the small intestine into the blood circu- lation; Boone as demonstrating passage into the portal circu- lation; Shuger and Arnold as presenting evidence of absorp- tion from the large intestine of exogenous bacteria; Timer as corroborating the last two works with yeast; Nedzel and Arnold as proving passage through the gall bladder; and Tqui as stating that bacilli invade the blood, bile, and urine through mucous membranes of the gall bladder, the invasion being made more powerful in cases of pathological change of the gall bladder (see bibliography "4" in the original for these references). Nedsel and Keller prepared their animals by fasting than for 24 hours. The duodenmn was exposed and tied off near the ‘ pylorus. Material was introduced into the stomach from the duodenum. B. prodigiosus was the organism used, and the blood was drawn from the right gastroepiploical vein at 5, lo, 15, 30, 45, and 60 minutes. They concluded: 1) that exogenous bacteria are absorbed from an isolated stomach into the blood; 2) that the greatest number are absorbed in the first fifteen minutes after introduction into the stench; and 3) that the lower the pH of the stomach contents the higher the absorption of bacteria. A. Seen: (5) in a study of the permeability of the guinea pig's intestine found that 30 to 50 days were needed for allergy to tuberculin to develop after one to five mg. of virulent tubercle bacilli were given by mouth. Ten to twenty milligrams of Bacillus Calmette-Guerine caused the animls to become sensitive to tuberculin 50 to 80 days after infection. Boquet, Voltis, and Scene (6) believe that the invasive- ness of the tubercle bacillus depends more upon the fatty structure of the bacillus than upon its pathogenicity as the grass bacillus invades in much the same manner. Hirsch ('7) in a recent paper discussed tuberculosis of the testicle. He quotes Oberndorfer as saying that in the tubules of the testes, as in other epithelial-lined canals, the tubercle bacilli presumably at first stimulate reactive changes imnediately beneath the epithelium, and then in the epithelium. The initial changes within the membrane propria may be slight, purely exudative, limited to a few round cells, and not distinctive. A hyperplasia of connective tissue cells follows promptly. Finally the canal lumen becomes filled with masses of desquamated epithelium, round cells, leucocytes, or, gradually, granulation tissues. The epithe- lium, at first lifted up from below, is more and more com- pletely destroyed, and the tuberculous inflammtion for some time is held in restraint by the elastic fibers of the basal layer. This, too, is gradually destroyed. ' s. a. Cullinan (a), in a case of tuberculosis of the stomach (apparently primary in this organ), found no giant cell systems. There were sparse accumulations of histiocytes and wandering mononuclears. Many of the necrotic foci con- tained very many tubercle bacilli. Stewart (10) divides intestinal tuberculosis into two important varieties; 1) secondary - usually a complication of pulmonary phthisis; and z) hyperplastic - apparemly a pri- mary infection. The hyperplastic variety is essentially pm... liferative, and ulceration, mile often or even usually present, is not as a rule tin conspicuous feature that it is in the secondary type. The walls of the affected portion become greatly thickened and hardened, all coats being involved. He finds that hyperplastic, or primary, tuberculosis of the intestine is a disease of the ileocecal region and usually confined to the last foot of the ileum in accordance with other opinions concerning the susceptibility of this level to disease. The ileum and cecum were also found the portal of entry in fifty-one patients with a primary intestinal focus by McIisnka. The intestinal lesion heals completely and is usually not desonstrable. The lymphoglanduler complex can usually be seen in multiple mesenteric lymph nodes and re- sembles exactly that of a primary pulmonary focus. In 2,500 autopsies 470 were found with residues of a primary intestinal infection. ‘ Post mortem observations on infmts who died in Lfibeck (18) after immunization against tuberculosis showed in eleven infants (out of fifty) the primary lesion in the small intes- tine. Next to this the primary focus was found most frequently in the tonsils and middle ear. Unusual primry locations were named as being the duodenum, esophagus, and stench. All cases of tuberculosis in children (intestinal) noted by Blacklock (13) in which ulceration occurred were fatal while city 71 % of 85 patients without ulcers died. By the time ulceration occurs there is usmlly a chronic catarrhal inflammtion of neighboring parts of the intestine (Ksrsner, 14). Lymphatics of the intestine around an area of ulceration of the mucosa show tubercles of lymphatic vessels particularly well. The conclusions of the Flinns (9) are in disagreement with the generally accepted conception that the polymorphc- nuclears are the primary aggressors in tuberculosis. According to them, tissue reactions to the tubercle bacillus are princi- pally manifested by the monocyts, the lymphocyte, and the neutrophil. The first sets as the primary phagocyte, later yielding the epithelioid cell and fencing the greatest part of the primary tubercle. It is not so conspicuous in healing as is the lymphocyte but is a scavenger of inflammatory products. The neutrophil is only outstanding in tuberculous suppurat ion, according to them. They find all of these reactions accurately represented in the blood count. Opis (15) gives an excellent general review of the present conceptions of the histology of tuberculous foci. He mentions the influencing factors of any inflammatory reaction as being: 1) the tissue in which it occurs; a) the permeability of the blood vessels in the different parts of the body; aid 3) the capacity of the tissue involved to permit exudation, continuing that with due regard to the peculiar characteristics of the tubercle bacillus as an irritant the nature of the reaction differs in various organs md tissues. His example is that of tuberculous pneumonia, an emotive lesion with charac- teristics reproduceable only in pulmonary tisaie. He objects to the division of tuberculous lesions into exudative and productive as being confusing and thinks that these terms should be used in a descriptive sense only. In early tubercle formation there is an emigration of polymorphonuclear leuco- cytes and other exudations from blood vessels. Mononuclears that accumulate in the alveoli multiply by mitosis and sssmne characteristics of those seen in the tubercle. The epithelioid cells may be derived from wanderixg calls of tissues or from blood cells, and some still claim endothelial origin. Hence the lesion cannot be called productive merely because of pro- liferation at the site. The affected part of a lung fourteen days after inocula- tion is described as unified by reticular fibers and accumu- lated cellular elements. Epithelioid cells are abundant. The reticulum is coarser at the periphery of the tubercle, where it is continuous with fibers of adjacent normal alveolar walls. Later these fibers permeate the whole tubercle. The appearance of collagenous fibers tending to replace the tubercle are con— sidered a continuation of this same process and a reparative phenomenon. He further states that any inflammation is charac- terized by exudation of fluid and emigraticn of pelymorpho. nuclears followed by an accumulation of macrophages. Bends is quoted as saying that an initial polymorphonuclear reaction occurs in man as in other animals on fomation of tubercles in the glomeruli of the kidney. Opie refers to Lurie's findings of more neutrOphils in rabbits after infection with the human bacillus, and more in the lung than in the liver. They were more abundant in tin more susceptible organ and with the more virulent organism. Lurie (16 and 1'7) deals with the correlation between the histologic charges and the fate of living tubercle bacilli in the organs of tuberculous and reinfe cted rabbits. Lurie used human and bovine strains as well as a culture of B C G brought back to virulence by cultural methods - a claim of interest in view of the continual controversy about the pathogenicity of that organism. In an attempt at quantitative determination of the tubercle bacilli a general parallel between the number of colonies isolated and the number of stainable bacilli was found, although cultural methods were necessary to determine quantitative relations. Acid fast material ranging from granular debris to ovoid or globular bodies was observed in cells. This is interpreted as consisting of fragnents of acid—fast bacilli and as possibly indicating destruction of tubercle bacilli. He found that the intravenously injected organisms were distributed in organs in the suns order in which parti dilate matter accumulates, i.e., spleen, liver, lung, bone marrow, and kidney. This quantitative relationship, however, is not maintained due to the greater efficiency of the mononuclears of some organs in destroying the bacilli. The most rapid growth of micro-organisms seemed to occur in these tissues in which the most extensive formation of new mononuclears was observed. The monocytes of the liver, splenic pulp, and bone marrow were found to destroy the bacilli more readily than those of the lung, kidney, or splenic corpuscle. Bacilli were less readily destroyed in the alveolar cavities than by inter- stitial monocytes. Less mitotic figures am the appearance of giant cells are correlated with the cessation of multiplication of the organisms, and conversely, multiplication and accumula- tion within mononuclcar leucocytes is found accompanied by active new fomation of these cells. This intense fomation and transformation into epithelioid cells Lurie finds con- stantly associated with destruction of the tubercle bacillus, in contrast to Sabin's contention that the "clasmatocyte" can efficiently destroy tubercle bacilli but that the monocytes and their derivatives cannot. The human type bacillus and the B C G, being the less virulent strains, are usually effectively destroyed'within epithelioid cells of all organs. In the lungs and kidneys the bovine bacilli persist within epithelioid cells. The giant cells appear earlier and in larger numbers in those organs that first destroy the bacillus (human and bovine bacilli and B C G). Immediately after infection accumulation of the less virulent types of tubercle bacilli do not cause caseation, and the more virulent bovine type produces this change only in the lung. Later caseation occurs in the presence of a small num— ber of bacilli and must be thought of as due, in part at least, to sensitization. It should be remembered that Lur ie's conclusions were based on the study of rabbits and not guinea pigs. An interesting statement in his report on experiments with reinfected rabbits is that an epithelioid cell may be described as a mononuclear phagocyte that has killed and incompletely digested tubercle bacilli or their products. Vorwald (18 and 19) has studied the early cellular reactions in the lungs of rabbits to the human type of tubercle bacillus. The injection was intravenous. The first cells to respond were the polymorphonuclear leucocytes, and the reaction was first confined to the small capillaries and, from these extended to the adjoining alveolar walls and spaces. 0 neu‘f‘f’ophsles, This intracapillary reaction, «thougx transitory, Vorwald con- siders of importance because it is through the agency of this cell that the site of subsequent development of the tubercle is determined. These cells phagocytose the tubercle bacilli in the blood stream and stagnate in small accumulations in the capillaries where the current is slow. In this way the injected bacilli are concentrated and localized. There was no . selective localization in the lymphoid aggregates (of the lungs). Almost always the bacilli lodge in the alveolar walls and then set up an inflammatory macticn. One hour after injection the neutrophils are found to form localized collections in alveolar capillaries. It is already difficult to find tubercle bacilli not taken up by them. This accmnulation is most evident fourteen to eigiteen hours after inoculation. At this time mononuclear phagocytes appear am ingest the leucocytes containing the tubercle bacilli, so that at twenty-four hours the focus of cells is predominantly mononuclear. Vorwald finds evidence here for the hematogenous origin of these reacting cells in the lack of mitotic figures seen. . Long, Vorwald, and Donaldson have worked on the mactions in the testes of normal and immunized guinea pigs to virulent human bacilli. In each series the importance of the polymor- phonuclear cell was shown. They conclude that the most important factor in the destruction of the tubercle bacillus is evidently the young epithelioid cell, but the polymorpho- nuclear leucocyte plays a part in preparing a way for it . (20) Feldman (21) produced tuberculous infections by various procedures in chickens, rabbits, guinea pigs, and dogs with the use of human, avian, and bovine strains. A comparative study of the anatomic character of the cellular reaction was made to determine whether its characteristics varied with the type of tubercle bacillus used to incite tin lesionsor whether the histologic character in a given species was constant regardless of the form of tubercle bacillus present. He con- cluded that the histologic nature of tuberculous infection in a given species is essentially the same regardless of the . origin or type of the bacillus responsible - so much so that he believes it injudicious to designate a given infection to be of human, bovine, or avian origin in the absence of tests for pathogenicity. He quotes Fox as saying that the peculi- arities of an order, family, or genus must play a very large role in the inception, development, and result of a tuberculous infection. ' In the liver and brain the monocytic cells appeared in the earlier stages as compact, diffuse accumulations with very irregular outlines- In the spleen the lesions in every instance had their inception in the Malpiguan bodies, where they expanded peripherally without demonstrable evidence of encapsulation. The appearance of giant cells of the Langhan‘s type was so inconstant in the guinea pig material studied "as to make it difficult to interpret the role of this cell in the tuberculous reaction. The three types of the tubercle bacillus gave similar lesions in the guinea pig as in the other test. animals. The cellular reaction in tuberculosis of the cornea has been discussed by Long and Halley (22). he initial response was on the part of the polymorphonuclear leucocytes. The fact that large monocytic cells became prominent only after vascularization had occurred caused them to experiment by placing one dose of tubercle bacilli close to the limbus and another dose in the center of the organ. One day after, the reaction was almost exclusively of polymorphonuclears in both. When vascularization had not yet reached the center, the reaction was mononuclear at the margin and polymorphonuclear at the center. At six weeks, the center was vascularized, and the reaction was mononucle ar at both places. Mitotic figures in the adventitia of vessels indicated an origin from this source also, as well as from the mononuclear cells of the blood stream. On intracorneal and subcutaneous injections of different amounts of tubercle bacilli (with rabbits and guinea pigs as test animals) different degrees of reactions were obtained (Santo - 24 and Schmincke and santo - 25), but the quality of the histologic picture was found to be the sme. Secondary lesions in regional lymph nodes were without relation to the number of infecting tubercle bacilli and were always maximal reactions. In human pulmonary tuberculosis it as found that the quality of the histologic picture was independent of the number of organisms demonstrated in a lesion. A rich primary implantation in the lung my cause a tuberculous pneumonia and not tubercles (Karsner). Duets contaninated with dry bacilli offer but little harm to healthy respiratory passages, but infection may be easily affected by the inhalation of fine liquid droplets con... taining the tubercle bacilli. Intratracheal inoculation of moist material brings excellent experimental results (Calmette). Fried (26) notes the reaction of the lungs at one minute to tracheal injections of mycobacterium tuberculosis as being a slight proliferation of lining cells of the alveoli. Cells lying free in the alveolus were often filled with acid—fast rods. The alveolus contained no serum, fibrin, or cells. After five minutes in many cases the air sacs were almost completely filled with cells possessing the characteristics of epithelioid cells. There was no evidence of activity on the part of capillary endothelial cells. At one week.the primi- tive tubercles had united to form areas of tuberculous granu- lation tissue with caseous centers. Fried believes this to indicate that the primitive tubercle is essentially intra- alveolar, resulting from a proliferation of alveolar epithelioid cells, which he considers mesenchymal and not epithelial. Following intratracheal infection the focus is more of the multilooular type than after infection by inhalation. Small, single foci conglomerate to one focus. The evolution of these foci is less regular than those produced by inhalation infection. Themononnclears mononuclears CI. day a neutrophils at neutrophils 8c neutrophils to neutrOphils c present a few rare none d {present present very numerous present e present very numerous present absent f absent absent present present g absent absent absent absent 3 a ¢ 20 37 15 18 b mononuclears mononuclears a mononuclears mononuclears b da. 1a neutrophils neutrophils & neutrophils a neutrophils c a few in lumen a few in a few in a few crypts crypts d present a few 1present present e a few a few absent absent f absent absent slight slight g absent absent absent ‘absent 5 a 30 35 37 43 b mononuclears mononuclears mononuclears mononuclears 3 da. & neutrophils & neutrophils &.neutrophils & neutrophils 0 present present present present d present present present absent e present present present present f absent absent absent present g absent absent absent absent 6 a 20 34 20 16 b mononuclears mononuclears mononuclears mononuclears 4 da. &.neutrophils & neutrophils & neutrophils & neutrOphile c present a few a few \~ 5‘, . . z 10!. set :31: 1.1.!lf O..|-r¢- ":5“ egg... -rll..rnr.§s.e.rrbn ..:.\ “it!“ . 1 V ...:-sn-....v:. fl 6 n r e O B .m a r m 0 K 15,000 12,600 5,600 Burnett: WHITE CELL COUNTS Full‘lltb.nessw2F,‘I1-mllee.mllfie uh!u~..’,nun,IU~N-HI-I~ I. ‘4.N.7.~vl.1—v-.‘u\ .s..~ ‘~._~ DIFFERENTIAL LEUKOCYTIC DETERMINATIONS Iniiiiiiion Gr°uP I I Group II 1 ' Pig No. 1 ' 2 ‘ 5 1 5 ‘ 6 4 ‘ 7 9 e ’ 15 Poly. 54 52 44 55 55 5O 55 56 59 59 Lymph. 59 64 54 5O 48 58 55 59 59 55 Mono. Eosin. 6 5 ll 11 8 5 2 4 Baso. 2 4 4 l l 2 Transit. l 1 2 2 l l l Eiggulzifgg 4 hr. 24 hrfl 2 day 5 day 4 day 5 hr. 24 hr. 2 da. 5 da. 4 da. Poly. 15 14 59 21 42 57 55 56 Lymph. 82 85 5e 77 55 62 65 53 Mono. 1 Eosin. l l 5 l 5 l 1 1 Base. 1 l l 1 Transit. 1 F‘“"§5fbre Inoculation Group III Pig No. 1 5 2 4 5 s 11 a 9 10 12 15 Poly. 24 52 42 26 25 5O 27 25 45 18 57 22 §;:§§ LyEPh' %§) 69 5:) 61 53) 55 6:) 72 7g) 75 5:) 66 $3) 67 ii) 75 43) 4s 7:) 76 5%) 61 73) 77 Mono. 5 2 l 2 l 5 5 2 4 5 l Eosin. 2 5 1 1 2 2 Baso. 2 2 l l 1 3 1 2 giggu12£fgi 6 hr. 1 da. 2 da. 5 da. 4 da. 5 da. 9 da. 11 da. 15 da. 19 da. 25 da. 29 da. Poly. 59 49 51 25 10 29 21 9 22 22 2O éngi Lyfph’ 5:) 57 4:) 48 5:) 64 7:) 74 7:) e5 6:) 68 ii) 76 $2) 88 :3) 74 :2) 78 :2) 78 Mono. 2 l 4 l 5 l 5 2 l 2 Eosin. l 1 l 1 2 Baso. 2 1 1 1 DISCUSSION The hemoglobin values obtained prior to death were almost consistently higher than those obtained before inoculation. However, there is no corresponding increase in the number of red blood cells to be noted. On Us contrary, there seemed to be more of a tendency for the red cell count to be lowered than otherwise. The white cell counts fluctuated a great deal. In all pigs (excepting numbers 1 and 5 of the third group) the percentage of lymphocytes was increased after ino— culation. While counting differentials of the second and third groups certain cells were noted, seemingly lymphocytes, with varying sized inclusions. In some cells these gave the appearance of blood platelets; others seemed like large granules; still others were single, homogeneous masses. It was difficult to determine whether these inclusions were basOphilic or acidophilic because of the intensity with which they took the stain. The following are camera lucida reproductions of some of these cells from group III. CONCLUSIONS Study of this series of experiments scans to indicate that tubercle bacilli of the human or bovine type in the lumen of the intestine do not possess sufficient attraction for leucocytes to cause their appearance in the lumen. Those few lacocytee which were seen in either the crypts or lumen did not contain any of the organisms. Moreover, the passage of the bacillus through the epithelial membrane was accomplished without the aid of wandering cells in the sections studied. This does not necessarily indicate that such a passage never occurs, but only that none of the lance cytes present in the epithelium were ever seen to contain mycobacterium tuberculosis. It is probable that many of the organism seen in the stroma and in the cytoplasm of epithelial cells of pig '7 (killed after 6 hours) are due to faulty technique, nevertheless sufficient observations of definite character indicate that the tubercle bacillus either unless its way into or is taken up by the epithelial cell and so affects its. entry into the tissues of the animal. APPENDIX APPENDIX 1‘- Ae a result of the failure to find organisms in the intestines of the first and second groups of animals it was decided to make measurements of the inte stinss of the third group to find if any reaction could be noted in this way. The average height of the longest and of the shortest villus was recorded as the height of the villi in a cross section of the intestine, and this was determined for each cross section of a level. The average of the broadest am of the shortest diameters of a lumen was taken as the diameter of that lumen, and this was determined for each cross section of a level. The average height of the epithelial menbrane was noted. Since pig 18 contracted tuberculosis of the intestine, this procedure was followed for all levels of this animal and for the first and second levels of pig 1 (6 hours), pig 5 (1 day), and pig 2 (2 days) also, at which time the procedure was discontinued as being of no value. The remlts for the first twolevels only of these pigs are given in the following table. The unallest height noted for any epithelial membrane was 19 u. (pig 12), and the greatest height for any manbrane was 57.8 n. tpig 3). II— In the discussion of to third group no mention is made of the colloidal carbon which was given to some of these pigs. None was seen. Moo oao mesa men some new «man ace .3 one 33 can A $3 «2. seed see one own new mos smog «as was man coo use «was new «as mum . Vsasm was seen coo some Ham ”Ham Hem coma oHo coma see when one use» awn n new moo» non new“ mne mesa mes send ”so men was a w _ «and one send , cam n. one one «saw «no coma use sees «as ”sad «as use” mom «mod «as «and use ‘ «and see coma use once one mean ”on one” wee oooa nae n.neaa use has» one new” use swam nan seed ems . one” new and» saw has” ems dead can use” «as amoa ode moan «as sees «so mama can i a m ; a..::H “to - H . a “0:“ ,e , Hp .. .n. ml: 9 . H IHP f a . m 413') , H... H H» He ~38.” a 962m 3333 23% 8:83 Sufism ~38an £30m H33 . once an -,na mam _ uses a u m a m , use H u n mam \t_ mason o - a mwm::_ 3:93.? a: HHH macaw ho mMZHamMHZH no mazn:nmnm4nznAdonaoqoamHm 1. 2. 3. 4. 5. 6. 8. 9. 10. BIBLIOGRAPHY T. B. MaGath and W. H. Feldman. Cultural methods and guinea pig inoculation. Am. Jour. Clin. Path., 2(3). E. T. Hellman and J. F. Witter. Some observations on the pathology of Johne's disease. Unpublished article, p. 12. A. Calmette. Tubercle bacillus infection and tuberculosis in.man and animals. 1923 edition, p. 121, 125, 134, 143, 146-149, 157, 162-163, and 277. A. J. Nedzel am R. Keller. Passage of exogenous bacteria through the wall of an isolated stomach. Proc. of Soc. of Exp. Biol. and Med., 30(6), 709. A. Soenz. Permeability of intestines in guinea pigs for virulent tubercle bacilli and for bacilli in the B C G 'vaccine. Ztschr. r. Tuberk., 56:131, 1930. Abst. in Arch. of Path., 10, 810, 1930. A. Boquet, J. Valtis, and A. Soenz. Mechanism of experi- mental tuberoulous infection. Ann. Inst. Pasteur, 46: 373, 1931. Authors conclusions - Arch. of Path., 13, 1002, 1932. E. F. Hirsch. Diffuse intratubular tuberculosis of the human testicle. Jour. A. M. A., 100, 15, 1160, 1933. E. R. Cullinan. A case of tuberculosis of the stomach. Jour. Path. and Bact., 33(2), 1013, 1930. J. W. Flinn and Z. M. Flinn. Tissue reactions to tubercle bacillus. Abst. in Jour. A. M. A., 100(18), 1466. M. J. Stewart. 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Jour. of Bact., xxv(2), 157, 1933. 42. S. H. Burnett. The clinical pathology of the blood of domesticated animals. p. 50. 43. J. Kolmer and F. Boerner. Approved laboratory technic. p. 65. 44. Max Pinner. Atypical acid-fast orgnisms. Proc. of Soc. for Exp. Biol. and Mad” 30(2), 214. 45. Herbert Fox. Disease in captive wild mmmals and birds. p. 485. 46. H. 0. Wells and E. R. Long. The chaistry of tuberculosis, 2nd edition, p. 139. AC KN OWLEDGMBNT I wish to express my appreciation to Dr. Hellman for his courteous assistance in all matters; to Dr. Stafseth for the bacillary cultures; to Dr. Chamberlain for the use of his camera lucida; and to Dr. Line for his willing aid in operative procedures. ”I? 1933 fig, ROOM USE ONLY, M|CHI AN STATE UNIVERS TY LIBRARIES G llllllllll lilllll II I I 015 6862 3 1293 O3