a. iio 3 1293 01589 LIBRARY Michigan State University VIO ( ~_ CF > Y OX PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES retum on or before date due. DATE.QUE DATE DUE DATE DUE — |-— == MSU Is An Affirmative Action/E qual Opportunity Institution STUDIES ON THE PATHOGENESIS OF THE AVIAN TUBERCLE BACTERIA. Thesis for the degree of DOCTOR OF VETERINARY MEDICINE by Lawrence A. Mosher. 1915. RACTERIOLOGY DEPT, As evidence of due honor and thankfulness for the assistance and directions of Dr. L. R. Himmel- berger and for the helpful criticisms of Dr. Ward Giltner, I express my obligations. 419937% STUDIES ON THE PATHOGENESIS OF THE AVIAN ; TUBERCLE BACTERIA. In December, 1913, Sir John M'Fadyean (1) of the Royal Veterinary College, London, England, completed a series of experiments relating to the vaccination of cattle against tuberculosis. Previous to his experiments Von Behring (3) showed that a degree of immunity could be produced in calves against tuberculosis by the intravenous injeotions of attenuated cultures of human organ-— isms, that had been grown for a mumber of years on artificial media. Unfortunately, this period of immunity was found to be one of short duration, and in order to successfully combat tuberculosis by this method it would be necessary to make yearly vacoinations. Later this procedure was found to be more or less dangerous in as much as the organ- isms were in some cases continually heing given off by some of the animals, more especially in the milk. Review of Literature. The work carried on by M' Fadyean was to deter- mine the efficiency of, intravenous injections of avian organisms, for immunization of calves a= gainst tuberculosis. In addition to the injeo=- tion of the avian organism, injections of the human bacterium were made for comparative pur- poses. In his work he assumed the avian organism to be nonpathogenic to the bovine species as far as producing any serious disease was concerned. But unfortunately, from a scientific point of view, he did not conduct any control experiments to show whether or not a tubercular condition could be produced in calves by this method of vacoination. He first grew the tubercular organism on five percent glycernated broth. The surface growth was removed and weighed, then injected intravenously into calves, in doses ranging from 1050 milligrams. The calves were at least three months old before injections were made. Aftera period of a few months, the calves were injected intravenously with 3-4 mg. immunity of tuberole bacteria of bovine origin to see if an immunity had been produced. -It was found that 3-4 me. of the bovine culture Was too severe a dose causing death unless the animal was killed in a morbid condition when at the point of death. The re- mainder of the calves, with the exception of two, Were injected suboutaneously with varying doses as it was thought that the immunity could be more accurately measured by this means. These two were injected 9 months after the first inoculation with 3 mg. of tuberole bacteria. In a large percent of the calves the subou- taneous inoculation had no effect other than pro=- ducing a local lesion at the point of injection. At times it was noticed after the injection of the avian vacoine, that some of the animals showed an elevation of temperature and often a cough devel- oped which usually lasted for only a few days. After a period of about a year the animals were killed and in most cases slight lesions were found in some of the organs. Whether this con dition was due to avian vaccine or to the injec- tion of the bovine culture is a question worthy of the utmost consideration. Series I. Record of M'Fadyean's Experiments. In this series eleven calves were used which were injected with the bovine tubercle bacteria on the 9th of Feb., 1913. Nine of the calves were injected with either human or avian organisms as. the table will show, while the remainder were used as controls. Series I. Tabulations of Injection. 13 15 16 30 56 ll 19 35 59 10/8/11 Avian 10 2/11/11 10/8/11 Avian 10 3/11/11 10/8/11 Avian 10 2/11/11 10/8/11 Avian 10 _ Controls (unvaccinated) 10/8/11 Human 10 ___ 10/8/11 Avian 10 2/11/11 10/8/11 Avian 10 2/11/11 10/8/11 Avian 30 _ Control (unvaccinated) Tabulations of Injections, (continued). No. Nature Dose Date of Dose Date of of bact. in mg. test inj. inmg. death. 8 __ 9/2/12 3 15/3/13 132 Avian 20 9/3/12 3 26/3/12 15 Avian 30 9/2/13 3 36/3/12 16 Avian 30 9/38/13 4 37/3/12 20 Avian 40 9/2/12 4 36/3/13 56 9/3/12 2 25/32/13 6 __ 9/2/13 5 23/7/12 11 Avien 20 9/3/12 5 8/13/13 19 Avian 40 9/3/12 10 33/7/12 25 __ __ 9/2/12 5 33/7/12 59 9/2/12 5 33/7/18 *Calves 8, 12, 15, 16, 20 and 56 were injected intravenously. Calves 6, 11, 19, 35, 59 were injected subcutaneously. Series I. Tavulated results. No. Liver. Lung. Spleen. Kidney. Pleura. 8 12 15 16 20 56 11 19 85 59 oOo + O + & O Oo 4 + + + 4 0 0 Tepicaracum involved) 0 + 0 o Oo 8 Oo 3 4 + o Oo 89 9 0 0 0 0 0 + (over lung) $ a ~~ Series I, Tabulated results, (continued). Wo. Peritonetm. Lymph nodes. Condition. 8 0 Bronchial Poor Mediastinal 13 0 Mediastinal Poor 15 0 Mediastinal Poor 16 0 Mediastinal Poor 20 0 Mediastinal Poor 56 0 Mediastinal Poor 6 0 Mediastinal Good 11 0 Mediastinal PresCap. Pharyngeal Good 19 0 Bronchial Prescap. Portal MNediastinal Good 85 + Pharyngeal Bronchial Mediastinal Hepatic Good 59 4 Tracheal Parietal Pectoral Poor Series II. This series represents fifteen calves, five of which had been vaccinated with the human type of tubercle bacteria, ( ons once, and four twice), while seven had received two injections of the avian organism. Of the remainder, two were used as controls, which along with the vaccinated an- imals received test inoculations with the bovine tubercular bacterium on the 18th of April, 19138. Calf (44 a) was also unvaccinated and was used as @ oontrol on the and test inoculation, having been injected intravenously with bovine tubercular bacterium. Test inoculation 44 a was made in- travenously, the remainder were subcutaneous. Tabulations of Injections. Date of Rature Dose Date of Nature No. lst vac. of bac. inmg. snd vac. of bac. 10 10/18/12 Human 10 __ __ 37. 35/11/11 Human 10 15/1/13 Human 39 35/11/11 Human 15 15/1/13 Human 42 35/11/11 Human 20 15/1/13 Human 44 35/11/11 Human 20 15/1/13 Human 17 10/18/11 Avian 10 3/11/11 Avian 16 10/18/11 Avian 10 2/11/11 = Avian 23 10/18/11 Avian 10 2/11/11 Avian 23 10/18/11 Avian 5 2/11/11 Avian 47 35/11/11 Avian 20 19/1/18 Avian 50 35/11/11 Avian 20 19/1/13 Avian 54 35/11/11 Avian 2L 19/1/13 Avian 57 Controls (unvaccinated) _—«6BB Controle (unvaccinated) 44a Controls (unvaccinated) Tabulations of Injections, (continued). “ose Date of Dose No. inmg. test inj. inmg. Death. 10 18/4/12 5 3/18/12 37 20 18/4/13 5 13/12/13 39 25 18/4/12 10 34/7/12 42 30 18/4/12 10 25/7/12 44 30 18/4/12 5 19/6/13 17 30 18/4/12 5 3/13/12 18 30 18/4/12 5 16/13/13 33 40 18/4/12 5 __ 33 50 18/4/12 10 34/7/12 4? 30 18/4/12 5 3/6/13 50 40 18/4/12 5 16/13/12 54 50 18/4/12 10 35/7/12 5? 18/4/12 5 2/12/12 58 18/4/12 10 13/6/12 44a 2/1/13 1 25/1/12 ll Series II. Tabulations of results. Perito= No. Lung. Liver. Spleen. Kidney. neum. Pleura. 10. O 0 0 0 0 0 a7. O 0 0 0 0 0 59. O + 0 0 0 0 43. 0O 0 0 0 0 0 44. 4 + 4 0 0 0 (knee joint contained infected fluid). 17. O 0 0 0 + 4 viseral & viseral & parietal parietal 18. ¢ 0 0 0 4 0 22. (had not been killed). 25. O + + 0 0 0 47. 4 0 + 0 0 0 50. #4 4 + 0 0 0 54. O 0 0 0 0 0 . Controls. 57. O + 0 0 0 0 58. + 0 4 0 0 0 44a. + + + + 0 0 ‘series II. 12 Tabulations of results, (continued). No. Lymph nodes. Condition. Seat of Injection. 10. 27. 39. 43. 44. 17. 18. 23. 23. 47. 50. 54. 57. 58. 44a. Prescap. Prescap. Prescap. Prescap. Prescape no. bact. Prescap. Prescap. Pharyngeal Bronchial Mediastinal Hepatic. Bronchial Mediastinal Mediastinal Hepatic Tracheal. Bronchial Hepatic Tracheal Mediastinal Gastric Pharyngeal Bronchial. Bronohial Tlliac Mediastinal Good. Good. Fairly good Good. Moderate. Good. Poor. Fair. Good. Moderate. Fair. Good. Good. Poor. Poore Thickened. Thickened. Thickened. Thickened & gritty. Thickened & gritty. Thickened & gritty. Thickened & gritty. Thickened & gritty. Abscess. Pin head tubercle. Gritty material. Gritty material. Gritty material & tubercles. 13 Series III. Fight animals were used in this series, two of which received two injections of the human vaccine, and four of which received two injections of the avian vaccine. The remaining two were used as controls. Tabulations of injections. Date of Nature Dose Date of No. let vac. of bac. inmg. 8nd vac. __ 38. 35/11/11. Human. 10 19/1/12. 41. 35/11/11. Human. 15 19/1/18. 21. 10/8/11. Avian. 10 3/11/11. 48. 35/11/11. Avian. 20 19/1/13. 53. 35/11/11. Avian. 20 19/1/12. 55. 85/11/11. Avian. 20 19/1/12. 28. Controls (unvaccinated). 29. Controls (unvaccinated). 14 Series III. Tabulations of injections, (continued). Nature Dose Date of Dose Date of No. of bact. inmg. test inj. inmg. death. 38. Human 20 20/7/13 20 2/11/13. 41. Human 25 20/7/12 40 13/12/18. 81. Avian 40 20/7/18 20 16/13/13. 48. Avian 30 20/7/12 20 1/11/13. 53. Avian 40 30/7/12 40 31/10/12. 55. Avian 50 30/7/12 50 1/11/13. 28. 20/7/12 20 31/10/13. 29. 30/7/12 40 1/11/18. 15 “Series III. Tabulated resulte. No. Lung. Liver. Spleen. Kidney. Perito- Pleura. _ neum. 88. 0 0 0 0 0 0 4l. 0 0 0 0 0 0 Ble + + + 0 0 0 48. + 0 0 0 4 0 parietal 53. 4 + 0 0 0 0 55. + + + 0 0 + (diaphragm & pericardium involved). viseral & parietal 38. 4 + 0 0 0 0 29. ¢ + + 0 0 0 16 ‘Series III. Tabulated results, (continued). No. Lymph nodes. Condition. Point of injection. 88. Prescap. Prepect. Coeocal. - Very good. - Soft swelling. 41. Bronchial. Mediastin. Tracheal Mesenter. Presoap. - - Good. - Soft swelling. Sl. Prescap. Bronchial - - Fair. Swollen. 48. Prescap. Bronohial Mediastin. Pectoral - = Good. - = Swollen. 53. Prescap. - = Good. - Swollen & gritty. 55. Presoap. - = Good. - Enlarged & fibrous. 68. Bronohial - - Good. —- Enlarged & custard ike. 89. Presoap. Bronchial Prepect. Portal - - Good —- Enlarged & fibrous. 17 ‘Series IV. This eeries consisted of twelve calves, five of whioh Were injeoted with human, and five with avian vaccines. The remaining two were left as controls. Tabulations of injections. Date of Nature Pose Date of Dose Date of No. vac. of bact. inme. test inj. in mg. Death. 61. 30/3/13 Human 10 21/6/13 50——s« 33/18/13. 68. 30/3/13 Human 10 21/6/13 35 3/13/13 63. 30/3/13 Human 10 21/6/13 50 3/13/13 64. 30/3/13 Human 10 21/6/13 385 4/13/13 65. 30/3/13 Human 10 21/6/13 = 50 4/12/13 71. 80/2/13 Avian 10 21/6/13 50 4/13/13 78. 30/2/13 Avian 10 21/6/13 35 5/13/13 73. 30/2/13 Avian 10 21/6/13 50 = 5/12/13 74. 30/3/13 Avian 10 21/6/13 25 38/10/13 75. 80/8/13 Avian 10 21/6/13 50 5/13/13 88. Controle (unvaccinated) 31/6/13 35 35/7/13 83. 21/6/13 50 4/8/13 -Series IV. Tabulated results. 18 Perito- No. Lung. Liver. Spleen. Kidney. neum. Pleura. 61. 0 0 0 0 0 632. O 0 0 0 0 0 63. 0 0 0 0 0 (pericardium affected). 65. + + 0 + & + onentum vl. + t 0 0 + + 78. 0 0 + costal 736 0 0 + costal diaphragm 74. #4 8) 0 0 0 0 75. + + 0 0 0 + Controls. costal pericardium 83. 4 + 0 + 0 ¢ 83. 4 + + + 0 Series IV. 19 Tabulated results, (continued). No. Lymph nodes. 6l. 63. 63. 64. 65. 71. 738.6 73. 74. 75. 82. 83, Prescapular Tracheal Prescapular Prescapular Mediastinal Prepectoral Pharyngeal Prescapular Prescapular Bronchial Portal Prescapular Bronchial P. mediastinal Pharyngeal Popliteal Prescapular Post medias=- tinal Prescapular Prescapular Bronchial Mediastinal Tracheal Mesenteric Prepectoral Prescapular Prescapular Prepectoral Mediastinal Mediastinal BSpleenic Submaxillary Tracheal Mesenteric Condition. Moderate Moderate. Moderate Moderate Moderate Moderate Moderate Moderate Poor Moderate Very poor Very poor Point of injection. Enlarged, contain- ing pus. Enlarged, dry and caseous Filrrous enlargement. Enlarged, full of pus e Enlarged, thick skin. Enlarged, thick skin. Enlarged, thiok ekin and abscess. Thick skin. Thick skin and Caseous. Thick skin & caseous. Swollen & necrotic Swollen & necrotic 280 M'¥Faydean, in his conclusions, presented the following: By intravenous injections of avian tubercle bacterium, it is possible to confer on healthy Calves a markedly increased power of resistance to infection with the bacterium of the bovine type. Such a method of vaccinating young cattle a- gainst tuberculosis involves little or no risk to the animal. When vacoinatiom of young cattle against tu- berculosis is considered advisable, the avian bac- terium should be preferred to the human in order to avoid the danger of infecting human beings with the bacteria that are present in the bodies of the vaccinated animals and passed out with their milk. Himmelberger (3) of this laboratory, ina study of the transmissibility of avian tuberculosis, has been able to produce the disease in calves by feed- ing in milk macerated organs from a tubercular hen. He also tested the animals with avian tuberoulin and was able to get a typical reaction. BL Previous to this, he injected two rats, two rabbits and two guinea pigs subcutaneously with five cubic centimeters each of a potato broth cul- ture of avian tubercle bacteria. One month later all were killed and upon autopsy the rats, guinea pigs and one rabbit showed no lesions, while the other rabbit showed an abscess at the point of in- jeotion in which tuberole bacteria could be dem omstrated. It might be stated that the oulture Himmelberger used had been cultivated upon arti- ficial media for a number of years. He also tried to infeot rats by alternating pens of rats with pens of infected hens, but met with m success. Along with his other experiments he tried to infeot rabbits by injecting intravenously two cen- timeters of a suspension of macerated diseased or- gans. Upon autopsy the rabbits showed no lesions either microscopically or macroscopically. The virulence of the human compared with the bovine tuberole bacteria has been studied at dif- ferent times by a number of men and practically all have concluded that the bovine organism is the 82 more virulent. Park and Krumweidd (4) in an extensive study along these lines, tested the virulence of the two Organisms by making intravenous injections into both rabbits and calves. Since only certain phases of their experiments have a direct bearing on our work, we will review only those portions which we believe are closely related to our studies. The material used by them was usually taken from a growth on egg medium. It was removed by means of a sterile platinum spatula and transferred to smooth filter papers so as to remove all moist- ure. The weight of the mass was determined and the proper dilutions made so that om oubic centimeter of the suspension comained one milligram of the bacteria. Other dilutions were made so that om cubic centimeter of the suspension represented .01 milligrams. The rabbits used were weighed both be- fore and after injection, with the exception that those receiving the human injection were weighed once in the interim. Through their work the principal object they B35 had in mind was to produce progressive lesions and from their observations they were led to believe that lesions from virus of low virulence retro- gressed; the number of lesions was found to be greater in those cases that died shortly after in- jection, than those that lived for a long period. Second, they did not make microscopical examina- tions of all lesions that showed no gross lesions, in as much as they thought that it would not give them absolute proof, unless a routine examination was made of the negative organs of other animals, as controls. Thus the number and location of the lesions was their index. A generalized form of tuberculosis was produced in every case when .01 milligrams of bovine tubercle bacteria were injected intravenously. When using the human bacterium in the same amounts intravenously no disease was produced, never producing a general- 4zed form. When one milligram was injected there was no generalization and as well no progressive lesions. When still larger doses were used death sometimes ocourred but the lesions found could not be considered as the cause of death. B4 In a rabbit injected with .01 milligrams of bovine tubercle bacterium, the following lesions might be visible: Lymph Lungs. Heart & Spleen. Liver. Peri- Rib nodes. adjacent tone= marrow. pleura. um & intes— tines. auxillary. inguinal. bronchial. mediastinal. mesenteric. hepatic. spleenic. In cases of one milligram injections, a more acute type of the disease was produced; the general picture being the same but in most cases the gross lesions were small. In some cases it should be stated that death resulted from acute infection. In cases of injections of .01 milligrams of the human type of tubercle bacteria, localized forms of the disease predominated chiefly in the lungs and less often in the kidney. By injeoting one milligram of the material the lesions were found to be about the same, possibly in some cases a little more marked; some generalization BS was observed. From the results they obtained they were able. to set a standard in differentiating the human or- ganism from the bovine by their virulence on rab- bits, which was as follows: When one milligram doses of the bovine organ- iem are used; lst. There is a tendenoy for the bovine vi- rus to kill acutely without any signe of general- ization. and. If death is uniformly caused by a vi- rus in less than thirty days and there is an ab- sence of generalization in those dying in less than twenty days, the bacteria is bovine. Srd. If a rabbit survives for fifty to six- ty days with lesions only in the lungs or kidneys or even a few scattered lesions elsewhere, the vi- rus is of the human type. 4th. Ifa rabbit dies within the first thir ty to sixty days without showing the presence of generalization, it would be safe to draw the con Clusion that it was due to the bovine tubercle pacteria. 6 A time limit was also set, when small doses were administered, which is as follows: A rabbit surviving a milligram for sixty days or .01 milligrams for forty days and showing no Signs of generalization was considered to have been — infected by a human virus. If there was any tend- ency toward generalisation with a large dose the smaller dose was also used, In their summary they made the following state- ments: The rabbit is the best animal for testing vir ulence for the diagnosis of type. The intravenous incoculation of .01 milligrams of culture is the best method of determining these differences. ny virus that is capable of produoing progressive generalized tuberculosis with this dose of bovine type, the ex- tent of the lesions being most important, the time of death being less important. ll virusea inoa- pable of causing lesions elsewhere, other than in the lungs or kidneys or in both are of the human type. Chance localization elsewhere is only occa- sional and not constant, being ruled out because a bovine virue is capable of producing a generalized —_ —_ - ——. ™ ae ee ea a se t= a7 form of tuberculosis in every rabbit. Virulence for Calves. Muoh work has been done as to the virulency of the bovine and human organisms when injected into Calves, but there has been an extreme variation of results as a whole. In the work previously discussed, it would seem that the human and bovine organisms are each in a class by itself. The following review will show the effects produced when the same organisms are injected into calves, &ll calves were tuberculin tested before being used. In every case where a bovine culture Was in jected, a rabbi’ was given a dose from the same source, fifty milligrams of a culture being injected into the neck of the calf. Eighteen strains of the human virus Were used, and only two calves showed any signs of dissemina- tion of the lesions. One calf showed doubtful le- sions in the lungs which proved to be positive when injeoted into a guinea pig. Another calf showed typical lesions in the mesenteric lymph nodes, ma- 28 croscopically, but when material from them Was injected into guinea pige it failed to produce the disease. None of the lesions were extensive enough to be considered ag generalized. Fourteen strains from the bovine were used, eight of these being from human sources. Of these, six produced a generalised form of tuberculosis, re- sulting in death after a period of from twenty-three to seventy-three days. One aalf showed a wide- spread form, which produced no ultimate effeot upon the animal. Another was practically non-virulent which was shown by the control rabbi?. In regard to those injected with the bovine virus, similar re- sults were obtained. Four caused progressive tuberculosis resulting in death in 20-90 days. One caused very slight tu- beroulosia but sufficient to influence the health of the calf. One was known to be nonmvirulent but was injected to compare with the control rabbits. From their work with calves they summarized as follows: When calves are to be used for the differentia tion of types, early cultures should be seleoted, 89 there being one disadvantage to the calf method as the bovine culture gives small growth. Parallelism with the rabbit is very close. The Royal Commission on Tuberculosis (5) was instituted by appointments in 1901. The purpose of this commission was to report upon the following: 1. ye tuberculosis in man and animals one and the same disease? 8. Can man and animals be reciprocally in fected with it? 5. Under what conditions if at all, does the transmissibility of the disease from animal to man take place and what are the circumstances favorable or unfavorable to such transmissibility? In this report the firet and second of these were dealt with. Two parallel investigations were carried on at two different farms, using calves of the Jersey breeds from three weeke to five months old. In some cases adult animals of the same breed or of the shorthorn breed were used. ks to the source of the bovine virus used, 30 ‘thirty lesions from naturally occurring tuberoulo- sia in cattle were used. In introducing this virus into the cattle, two methods were employed; (1) feeding, (3) injecting into the tissue. Of the tissue injections, three methods were used, subcu- taneous injections, intravenous injections, and in- tra mammary injections through the teat opening. The effects produced by these injections may be summarized as follows: The bovine tuberoulo- eis bacterium when introduced subcutaneously into the body of the bovine, whether in the form of e muleion or as a culture may produce; (1) fatal tu- beroulosis of generalized form, (3) a limited retrogressive tuberculosis, (3) or may show inter- mediate effects. The amount of disease produced was found to rest upon certain factors, for example, the amount of material injected and the susceptibility of the animal. The work done with the human bacterium consis~- ted of securing material from sixty cases of human tuberculosis. Of these two groups were formed, being distinguished by the properties and character- ee eo ee eee 31 istios of the bacteria. A third group was formed which could not be classed under the first or sec- ond as it had properties of its own. Group I consisted of fourteen oases, the virus- es being obtained from either sputum or surgically removed tubercles from cervical glands, and primary cases of abdominal tuberculosis. All the cases of abdominal tuberculosis were obtained from ohil- dren. Group II consisted of forty cases of different origin. In some cases the material consisted of material removed surgically, eight from mesenteric lymph nodes removed after death, ten from lungs and bronchial glands, or from a diseased kidney, ons from a tuberoular testis and the remainder were taken from diseased joints or bones. The effect of the bacteria of the first group, when injected into calves was found to be the same as the effect of the bovine bacteria when injected into calves. The bacteria of group II, was found to be far lese virulent in large doses. It failed to produce progressive generalized tuberculosis in the bodies 33 of rabbits and calves. When subcutaneous injeo- tions were made it was found that the resistance of the body tissue was greater than the virulence of the organism, thus setting up a retrogressive form of tuberoulosis or none at all. Group III was not placed under this head be- cause the organisms produced like results when in- jected into animals but were grouped for the sake of convenience as they had individual character- istics. Their Conclusions. There can be no doubt that in a certain num ber of cases the tuberculosis ocourring in the hu- man subject, especially in children, is the direct result of the introduction into the human body of the bacterium of bovine tuberoulosis; and there also can be no doubt that at least in the majority of these cases the bacterium is introduced by the consumption of infected cow's milk. Cow's milk containing the tuberole bacteria of bovine source ie clearly the cause of tuberculosis of fatal ter- mination in man in many cases. Of the sixty cases of human tuberculosis in- 33 ‘vestigated, fourteen belonged to Group I, that is — to say, they contained the bovine virus. Instead of taking all forty of these cases, they confined themselves to those cases of tuberculosis in which the bacteria were apparently introduced into the body by way of the alimentary tract. Of the total eixty casee investigated, twenty-eight possessed Clinical history indicating that the bacteria were introduced by way of the alimentary tract. Where cervical glands were studied, a large peroent were found to be of alimentary origin. These facta indicate that a very large percent of cases of tuberculosis in man contracted by im gestion is due to the tuberculosis bacterium of the vovine source. A very considerable amount of disease and loss of life in man, especially among the young mst be contributed to the consumption of cow's milk har boring the tubercle bacteria from diseased bovines. The presence of the organism can be detected in the milk though with some difficulty if the proper means be adopted. Such milk should never be used as food. 34 They further state, that from a health stand-— point, more stringent laws should be enforced as to the sale and consumption of milk. Technique of Our Experiment. The cultures used were grown on glycernated potato and carrot slants. After the organism had grown luxuriantly they were removed with a sterile platinum loop and placed on sterile filter papers to dry. As nearly as possible all the moisture was removed. It was then weighed and the proper dilutions made in sterile salt solution so that one cubic centimeter of the suspension contained e001 gram of the dried avian organism. The rabbits used were all weighed before in- jeocting and again at death, so that the loss or gain in weight might be determined. There were a few rabbits injected with .01 gram of the sus- pension, but it has been our experience, as well as that of Park and Krumwiede, that when a large dose is injected there will develop an acute form of the disease and usually death will result before any macroscopic lesions develop. 35 In this series which we will designate as se- tries A, eighteen rabbits were subjected to intra- venous injections of a suspension of avian tuberole bacteria. Two were injected with a macerated diseased spleen of a hen, two with macerated bovine lesions, and two with a suspension of material from a human culture. Some of the rabbits were autopsied in thirty days, When in a moribund condition, while others were allowed to live for four months. If no gross leaions were found at death a microscopic examination of the material Was made. Tabulation of Results. Culture &No.daysLoss in Post Mortem Findings. No.amt. inj. lived weight. 1. N.D. .01 280 323 gms. No lesions found on GM. macrosocopical examina= tion. Many tubercle bacteria found micro- scopically in liver, lungs, spleen & kidney as Well as adjacent lymph nodes. 36 Tabulations of Results. Culture & No days Loee in No amt. inj. lived. weight 6. N.D .O1 28 410 Gms « gms . 3 Mixed Wis. 19 350 eOl gms. gms. 4 Mixed Wis.138 1180 -Ol gms. QMS « 5 Me. 01. 43 490 gms . gms. 5a 13 .001 109 715 gms. gms 4a 13 .001 106 785 gms. gms. 1375 .001 36 693 gms. gms . Post Mortem Findings. Lesions in apices of both lungs. Bac- terium microscopically in liver, spleen and adjacent lymph nodes. Tubercles in liver and spleen. Found bac- teria in lungs. Kid- neys normal. Very badly emaciated, hind parts paralized. No maocroscopical lesions. Bacteria were found in lungs, liver, spleen, kid- ney and adjacent lymph nodes. Femoral-tibial joint involved. Fair condition. Tuber- Gles found in liver, Spleen, and smears from lu showed many bacteria. Lesions in lungs, liver spleen, kidney. Perito- neum slightly involved. Lunge, liver, spleen and kidney badly involved. Parietal peritoneum studded with small tubercles in region of kidney & stomach. Both lungs badly involved with small tubercles. Spleen, kidney and adjacent lymph nodes, involved. | 37 Tabulations of Results, (contimed). No Culture & No days Loss in Post Wortem Findings. amt. inj. lived. Weight. _ 6 13 ,.001 144 565 Lungs, spleen, he~ QMS e gms. patio and mesenteric lymph nodes involved. 14 75 .001 Killed 787 Lungs present many GMs « on 114th gms. small tubercles. day. Spleen normal. Kidneys and soapro-humoral jointe involved. 15 135 .0021 63 793 Macroscopically, per=- gm « gms. itoneum, kidney, liver, Spleen; microscopically, Lungs. 19 143s .02 30 760 Mesentery, intestines, gms. gms. liver, spleen, kidneys and lymph nodes. $1 75 .01 29 1010 No macroscopic lesions CMS» gms e except in hepatio lymph nodes. Found tubercle bacteria microscopically in lungs, hepatic glands, kidney and spleen. 538 135 .0O01 46 310 Lungs, liver, spleen, gms. gme. kidney, all showed lesions. 18 Mixed 75 16 630 Liver, lungs, thoracic & N.D .001 gms. Wall and thoracic pleura, gms. parietal peritoneun, Spleen and lungs. 3a 13 .001 90 343 Lungs, liver, spleen, GMS « gms. mesentery and peritoneun. la 13 .001 72 470 Lungs, thoracic wall, EMS « gms. peritoneum, spleen liver. 58 Tabulations of Results, (contimued). ‘ No Culture & No days Loss of P amt. inj. lived weight. 23 A. Be De Ee F. 13 .001 114 348 gms . gms. gain. Bovine Killed tissue on 45th day. Bovine Killed tissue on 45th day e Human Killed culture om 60th e001 gms e day @ Human Killed on culture 60th day. Avian Killed on tissue 45th day. Avian Killed on tissue 45th day. ost Mortem Findings. No lesions macro- scopically or micro=- scopically. Milary tuberculosis of liver, spleen, kid—- ney, lungs and dia-~ phragm. Lesions inl 9 liver, spleen peritoneum. Lesions in the apex of left lung. No other evidence of infeotion. Several small tubercle fooi in both lungs. Lesions in liver, spleen and kidney. Lungs, peritoneun, spleen, kidneys and diaphragm. From these results it can be plainly seen that the rabbits receiving intravenous injections of avian tuberole bacteria developed a generalized form of tuberoulosis. The rabbits receiving the injections of a diseased macerated spleen from a hen developed a like form.of the disease. 39 The two rabbits receiving the injections from the diseased bovine tissue, developed a very severe form of generalized tuberculosis which resembled that from produced by the avian organisn. In the case of those injected with the human organism, a localized form was the result. Series B. This series consisted of fifteen rabbits in- jected with cultures of the avian organism from different sources. Two of the cultures were from the North Dakota and Wisconsin Experiment Stations, while the third was isolated from the spleen of a hen, by the author. They were grown on glycernated potato and carrot slants. In series A we had great difficulty in making some of the intravenous injections in as much as the suspensions contained 6o many clumps. In this se ries, we devised a method that worked more satisfac- torily and did away with all previous difficulties im injecting. The growth was removed from a slant as before, with a sterile platinum loop and placed between two sterile filter papers to remove all moisture. To 40 ~the mass was added a quantity of sterile NaCl. so- lution making a concentrated suspension. This was placed in the shaking machine which was allowed to run for about three or four hours. The suspen sion was then filtered through a small piece of cotton to remove all the large olumps so that it would pass easily through a fine hypodermio needle. This left a heavy, milky suspension which was Weighed. The weight of the Na.Cl. solution Was subtracted from the weight of the suspension which gave the weight of the bacteria. The proper di- lutions were made eo that each oubic centimeter by weight contained .001 gram of the avian culture or the dose for a rabbit. £11 rabbits injected were females, the injeo- tions being made intravenously in the right ear. Tabulation of Series B. Amt. of Loss in Wo days No.inj. & weight. lived. Autopsy Findings. culture Le 2001 570 88 Much emaciated liver, MS o gms. lungs, spleen and ad- jacent lymph nodes affected. ” 41 Tabulation of Series B, (cont imed). Wo.Amt. of Loss in No. days Autopsy Findings. inj. & weight. lived. culture } e001 770 58 Lesions in liver, gms. gms. Spleen, lungs. Micro- soopical examination of the kidneys showed numerous tuberole bac- teria. . e001 880 38 Liver, spleen and gms . gms. kidney affected. e001 715 48 Much emaciated lesions SMS « gms. found in the junge. liv- er, spleen and jacent lymph nodes. Kidneys showed a few pinhead Lesions. e001 590 39 Lungs, liver, spleen SMS gms. and adjacent lymph nodes affected. e001 400 70 Lungs, liver, and adja- gms. QMS « cent lymph nodes affeoted. No bacteria were found in other organs. e001 430 70 Lesions in lungs, liver, @ms. gms. spleen and adjacent lymph nodes. Kidneys showed no tubercular bacteria, microscopically. e001 1330 70 Very poor in coniition. gms. gms. Lesions in lungs, liver and adjacent peritoneum; spleen studded with large tubercles. 42 Tabulation of Series B, (continued). _ Yo.Amt. of Loss in No. days Autopsy Findings. inj. & weight. lived. culture grams. 9 .001 80 gms. 10 .001 1090 gms. 11 .001 1140 gms. 12 .001 170 QMS 13 =©.001 5 gms. gain. 14 .001 415 15 .001 500 76 Sl 37 71 84 killed. 41 54 Found slight lesions in lungs and liver. Microscopically, no bacteria were found in other parts. Much emaciated and un- able to eat; lesions found in lungs, liver, spleen and adjacent lymph nodes. Kidney showed many bacteria microscopically. Much emaciated; breath- ing labored at time of death. Autopsy showed a consolidation of a great- er part of both lungs as well as the parietal pleu- ra; liver, spleen and both kidneys showed many lesions. Lungs and liver showed a few lesions. Mediastinal lymph nodes involved; no bacteria were found in other organs, microscopically. In good condition, a few bacteria were found in the lungs. Fair condition; lesions in lungs, liver, spleen and ad- acent lymph nodes; microsoop- cally found bacteria in kidneys. Lungs, liver, spleen and adjacent lymph nodes were af- fected; condition fair; micro- scopically, no bacteria were found in the kidneys. 45 Series A. Tabulations of Parts Affected. Rabbits showing lesions or the microérganisns microscopically in five groups of organs. Ro eLungsLi-Spleenkid- Pleu-Per—LymphDia— Wesen- ver ney ra. ito-nodesphragm tery. neum +. + + + 0 0 + oO 0 (femoro-tibial joint) A +¢ + + + O o +¢ + ° Rabbits showing lesions or the microorganism microscopically in four groups of organs. L + + + + 0 0 + O 0 5a + + + + 0 + 0 0 Oo 4e0 + + + + O + O 0 O 15 + + + + ° + Oo ° O 32 + + + + Oo Oo O O 0 F + QO + + 0 + 0 + ° Rabbita showing lesions or the microorganism microscopically in three groups of organs. 18 + + + 0 + + O O O 2 + + + 0 0 Oo + Oo 0 Ss ¢ + + Oo Oo o.060OUCOO O 0 5 + + + O 0 QO 0 ° ° 13 + + + 0 0 Oo O 0 0 44 Series A. Tabulations of Parts Affected, (continued). No.LungsLi-Spleen Kid-Pleu-Per= Dia- Mesen- ver ney ra. ito-Lymphphragmtery. neumnodes 14 + oO + oO oO o o O oO (scapuloshumoral joint) 19 ° + + + 0 + + O oO 31 + + + oO oO O + oO oO 18 #+ + + 0 + + oO O oO 38 + + + 0 oO + ° Oo + La + + + ° oO + O ° oO E + + + oO + + O oO oO E ° + + + oO oO oO 0 0 Rabbits showing lesions in one tract. + D + 23 Non. infected, Series B. Rabbits showing lesions gr the microorganism in four groups of organs. 2 + + + + 0 oO 0 4 + + + + 0 0 + 10 + + + + o o + 11 + + + + + o + 14 + + + + 0 o.0OCUt 495 series B. Tabulations of Parts Affected, (continued). No.Lungs Li-Spleen Kid-Pleu-Per-Lymph Mesene ver ney ra ito-nodes Dia- tery. neum phragm Rabbits showing lesions or the microorganism in three groups of orgens. 1 + + + o Oo 0 + ° Oo 3 O + + + O O 0 O 0 5 + + + 0 0 O + O 0 ? + + + 0 O ° + Oo Oo 8 + + + 0 O + Oo O Oo 15 + + + 0 ° QO + 0 0 Rabbits showing lesions or the microorganism in two groups of organs. , 6 + + oO 0 oO O + oO 0 9 + + oO oO oO oO oO oO o 12 + + ° Oo 0 Oo + 0 o Rabbits showing lesions or the microorganism in one group of organs. 13 + 0 0 0 oO ° oO o oO +Indicates that this part was found to be ine fected either microscopically or macroscopically. 46 Assuming that when three tracts are involved a diagnosis of progressive generalized tuberculosis can be made, we oan say that we have produced the above form in 832.5 percent of the cases under our observation. In following the work of M'Praydean, some very interesting comparisons can be made of the patho- genesis of the human, bovine and avian tubercular bacteria. | In series I, calves 6 and 8 received but one injection of the human vaccine before being treat- ed with the bovine test injeotion. Both calves developed a very severe form of tuberculosis Which was detected on autopsy. Seven calves received the avian vaccine of which five received the second injection before being subjected to the bovine test ‘dnooulation. Upon autopsy it was found that they had all developed as serious a form as those injeot- ed with the human organism. The two controls were found to have a generalized form, being badly emaciated. In series II, there were fifteen calves used of which five were subjected to the human vaccine, 47 seven to the avian and the remaining three were used as controls. It would seem that the avian organism was far more virulent than the human organism when injec- ted into calves intravenously or that more protec- tion against the bovine organism was conferred by the human vaccine than by the avian. A large percentage of those receiving the avian vaccine were very seriously infected, having @& number of organs and tissues involved,while in the case of the five receiving the human vaccine only one was seriously affected and that was lo- calized in the lungs. The first control calves were in fair condi- tion, having lesions in the lungs, adjacent lymph nodes and the mesemteric lymphatios. The remain- ing two were badly affected, having developed a generalized type of the disease. In series III, there were eight calves used, two of which received two injections of the human vaccine, and four received two of the avian. The remaining two were control calves. The two receiving the human vaccine were in good health and showed only a few erall lesions in some of the lymph nodes. The four receiving the avian vaccine showed lesions in the lymph nodes and one or more of the vital organs, three of these being of the generalized nature. Both of the calves were in good condition, calf 28 having developed a localized form while calf 29 developed @& generalized form of the disease. This series would lead one to believe that the avian organism Was more virulent than the human and that there existed a relation as regards pathogenesis to that of bovine origin. Calves receiving human organiems, localized form 100%. Calves receiving avian organisms, localized form 325%. Calves receiving avian organsims, generalized form 75%. Calves receiving bovine test injection, generalized form 100%. (This is further evidence of the great virulence of the bovine type). Series IV. In table 4 there were twelve calves used of which five received one injection of the human vac- Gins, and five received one injection of the avian 49 vacoine. The remaining two were unvaccinated controls. Of those receiving injeotions of the human vaccine, three had slightly infected lymph nodes of different regions, one,infection of the lungs and adjacent pleura, while the fifth developed a generalized form. Of those receiving the avian vacoine, all but one had infection of some vital organ, while calf ll had a generalized form. Control calves 82 and 83 were both in poor condition and had a very severe form of generalized tuberculosis. From the foregoing it can be plainly seen that there was infection in more vital organs and more forms of generalized tuberoulosis by using the avian vaccine than by using the human. In our experiments we have found that a large peroent of the cases of progressive, generalized tuberculosis had lesions either in the lunge, liver or spleen, but often times showed other infected foci in the different parts of the body. Most of the cases of miliary tuberculosis, both in series A and B, were cases of short dura- 50 tion, death usually ocourring before there was much emaciation or any outward clinical symptoms. In series A, rabbit A, injected with material from a Giseased lymph node of a cow , was in very poor condition at death; but sinoe the weight of the material injected as well as that of the rabbit was not taken, an exact determination of the virulency Oan not be estimated. Of those in series B that showed an involvment of two tracts, six were considerably emaciated, while rabbits 9 - 12 were apparently in good health at death. Number 13 of series B was in good health at death (killed), gaining in weight during the experiment. It was found that those rabbits having an in- fection of five tracts died within a period of from twenty to twenty-five days either of an acute infection or miliary tuberculosis. In studying the effects of the different or- ganiems we found that there was no great variation in the virulence of the different cultures as to the number of organs affected but it wae found that Sl certain cultures would produce a diseased condition in a shorter period of time than others. In studying the different organiems as to their patho- genic effects the individual tolerance of the rab- bit must be considered, as for example, rabbit 33 in series A and 6, 9, 12 and 13 of series B did not develop a serious form of the disease when kept un- der the same condition as those whioh were in a more or lees morbid condition at death. | Description of Plates. The material used for sections was obtained from the rabbits of series A. A number of sections were made and part of them were stained with heated carbol—fuchsin, then counterstained with methylen blue, while the remainder were stained with hematox- ylin and eosin to show the pathologic changes that had taken place in the tissues. The type of the disease produced was found to be somewhat variable in the different animals and the pathological changes in the different organs were uniform. Ae Was previously stated, those animale receiv- ing large doses developed an acute form of the di- 53 eease and many times showed no macroscopic lesions at autopsy with the exception of now and then a hemorrhage of some part of the body. But in the rabbits that received the smaller doses there was no difficulty in finding lesions either in one or more of the organs. The animals will first show signs of emacia- tion and loss of appetite. In some cases diges— tive disturbances were noticed and as the disease progressed the lung disturbances were evident from the increased respirations. Those animals that died of the ohronic form of the disease showed in many cases the miliary form of tuberculosis, most noticeable either in the liver or spleen. Natural Infection may take place by one of the following methods: Ingestion, Inhalation or Wound Infection, but it is supposed that in most cases infection takes place by the first method. After entering the body the organiems are disseminated through out the body either by the blood or lymph streams and are finally deposited in the tissue from the capillaries. 55 The number of tubercle bacteria that can be found in the different lesions when examined micro- scopically, is extremely variable. The location of the organism is not always constant but depends upon a number of factors such as the organs affected and the extent and location of the lesion. Where there is an acoumulation of epithelioid cells the bacteria may be contained within the cell body, but if there is a considerable amount of cell necrosis taking place they may be set free and will be found in the intercellular spaces. They appear to grow and spread within the lesion by direot growth from cell to cell with the aid of the ameboid movement of the cell. Liver. It is quite evident that infection of the liver takes place through the hepatic artery and portal vein. The lesions may be located in any portion of the organ but are more often seen in the region of the portal vein. The chains of liver cells will be more or less destroyed by the formation of epithelioid cells, Figure I. Some of the cells may show the presence of necrosis. Figure II 54 shows a large ares of necrosis and the presence of | many tuberole bacteria. Spleen. Infection of the spleen takes place through the blood streams and it is often associated with generalized tuberculosis. Often times the organ will be much enlarged ani show areas of extreme hypodermia with an acoumulation of epithelioid cells which will be easily detected upon microscopical examination. The Malpighian bodies are the last to become infected but finally fail to resist the in vading organiems resulting in a proliferation of epithelioid cells. Figure III shows a section of the spleen studded with tuberole bacteria. Lungs. Infection takes place either through the blood or respiratory tract and in some cases the lymphat- ios. The type of disease produced may vary some- what. Acute miliary tuberculosis is one of the more frequent as well as serious forms. The tuber- Cle bacteria probably reach the tissue by way of the blood streams and in this way are disseminated throughout the organ. 55 In chronic tuberculosis the first lesions either arise from the blood or through the air passages, occasionally through the lymphatics. There will be first noticed a proliferation of epi- thelioid cells, later necrosis and in a number of cases calcification. Softening of a tubercle lesion is quite frequent, often times being due to the invading material. The softened material may discharge into a bronchus and produce caseous pneumonia or into a blood vessel and cause an acute form of generalized tuberculosis. Regeneration of connective tissue may take place around a tubercle due to the organization of the fibrin produced as a reeult of the inflamma- tion. Extensive fibrinous deposits are quite common in tuberoulosis lung. Figure IV. shows an extensive diseased area filled with necrotic lung tissue and endothelial cells. Kidney. The types of tuberculosis produced in the kid- ney are not always the same. The three following types are most often found: maliary tuberculosis; tuberculosis infarcation and tuberoulosis nephritis. 56 Miliary tuberoulosis is more often seen than | the other two forms, but it is not as frequent as miliary tuberculosis of the other organs. The le- sions may firet be seen in a glomerulus or in the Capillaries between the tuberoles. There will be noticed an area with an acoumulation of epithelioid Cells which later will change into areas of necrosis as the vessels are occluded. In cases of kidney infection the animal will soon die as it is quite likely that the primary seat of the disease is in some other organ. Tuberculous infarcation is a form of tuberoulo- sis often seen in the kidney and results from infeo- tion from an artery by which the tuberole bacteria are distributed to the smaller vessels of the main artery. Tuberculous nephritis is a more chronic form of tuberoulosis and is due to the organisms gaining entrance into the pelvis of the kidney thus produc- ing pyelitis. The tubules will later be infected leading to a spread towards the cortex of the organ. Numerous absoess formations will be found which will later result in necrosis and ulceration. 57 Lymph nodes. Of the lymph nodes most frequently infected are the cervical, mediastinal, bronchial, hepatio and mesenteric although others may be involved. Infection likely takes place more frequently through the lymphatics, less often through the blood. The bacteria are probably disseminated by means of the ameboid movement of the epithelioid cells. The organisms that are carried to the produce a generalized form of tuberculosis while those that remain in the sinuses cause an acoumula- tion of leukocytes which fill and destroy the vessel. The miliary lesions usually spread and in a short time will form one solid mass by the fusing of the numerous tubercles. Conclusions. It would seem unsafe to use the living avian organism in preparing a vacoine for cattle. It is usual to produce in rabbits a progres- sive form of generalized tuberculosis by intraven- ous injections of the avian tubercle bacteria. In larger doses than .001 gm. intravenously 58 an acute form of the disease will be produced causing the death of the rabbit in a few days. From the results we have obtained it would lead one to believe that a similarity exists between the avian and the bovine type of the tuberole bacteria as regards their virulency. Literature. (1) M'Faydean: Jour. of Comp. Path. and Therap., Vol. XXVI, Part 4 — Dec., 1913. (3) Von Behring: The Suppression of Tuberoulosis. (3) Himmelberger: Centralbl. f. Bakt. Parasiten. u. Infekt., 73 Band. 1914. (4) Park and Krumweidi: Jour. of Med. Research, Vol. XXV, pe. 3135. (5) Royal Commiesion of Tuberculosis: Jour. of Comp. Path. and Therap., Vol. XXIV. Finis. Fig. IT. 19377 ’ \erore?: aw ehsiDb aw 4 me “on 7s uo. ; « x ’ oy’? WoW LIAS NY 922 805.0 oaAynaecia SORA pres “Te bt >So. +? oO. er Ae eA ek ra + LL on ae BiLWIevrele RARIES MICHIGAN STATE UNIV. LIB UNNI 312930158954 71