MYCUBACTERIOSIS OF CATTLE ENOCULATED WITH GROUP-Ill ORGANISMS OF BOVINE ORlGlN Thesis for flu Degree of DE. D. MECHIGAN STATE UNIVERSE” Lloyd Samuel Goyin'gs 1965 IHESlS LIBRARY Michigan State University This is to certify that the thesis entitled MYCOBACTERIOSIS OF CATTLE INOCULATED WITH GROUP-III ORGANISMS OF BOVINE ORIGIN presented by Lloyd Samuel Goyings has been accepted towards fulfillment of the requirements for Pho Do degree in Path010gy QlL/ELZ/“c VJ? i y‘égt—L (71", awgfli/ Kr I J Major professor Date ‘/€M?~“ i"”}7 (,2- 5: / ?69 5 / 0-169 R0052“: 1552 GEM ROOM USE m ABSTRACT MYCOBACTERIOSIS OF CATTLE INOCULATED WITH GROUP-III ORGANISMS OF BOVINE ORIGIN by Lloyd Samuel Goyings Two experiments were conducted to determine the relative pathogenicity of Group-III mycobacteria of bovine origin in cattle when inoculated by the intrauterine route and by aerosol. The results were determined by tuberculin testing, serologic procedures, clinical observations and detailed macrOSCOpic, micrOSCOpic and bacteriologic examination of tissues. Intrauterine Experiment Nine heifers between 21 and 23 months of age were di- vided into three groups of three heifers each. Each group was inseminated with semen containing a different strain (1 mg. wet weight) of bovine-origin Group-III mycobacteria after artificial production of estrus. One week prior to each scheduled necropsy, all heifers were tuberculin tested in the caudal fold with mammalian tuber- culin and in the cervical region with mammalian and avian tuberculins and johnin. Following the tuberculin tests9 one Lloyd Samuel Goyings heifer from each group was examined post-mortem at 2, 4 and 6 months after inoculation. The six heifers, three inoculated with culture 510-0 and three with culture 680-0, had evidence of generalized disease. Three heifers inoculated with culture SOB-O had little evidence of disease. The distribution of the lesions indicated that the in— fection Spread from the uterus to lymph nodes via the lymph- atics and to the peritoneal cavity through the Opening of the fimbria of the Fallopian tube. Further spread of the infec- tion occurred from the peritoneum to lymph nodes via the lymphatics. The heifers examined post-mortem at 4 months or at 6 months after inoculation (except those inoculated with culture SOB-O) had evidence of both lymphatic and hemato— genous Spread of the infection to other organs and lymph nodes. The lesions found in the lymph nodes of the head region suggested oral reinfection. There was a marked dissimilarity between the distribu- tion of lesions observed in naturally acquired bovine tuber- culosis and the lesions found in the heifers inoculated by the intrauterine route with cultures SleO and 680-0. Lloyd Samuel Goyings Group-III mycobacteria indistinguishable from the strains inoculated were recovered from most of the lymph node pools which had lesions. Frequently isolations were made from lymph nodes with no detectable gross or microscopic lesions. Heifers inoculated with culture SlC-O had a progressive decrease in tuberculin sensitivity after an initial hyper- sensitivity. There was a negative correlation between the results of the tuberculin sensitivity and the presence of gross and microscopic lesions. In contrast, heifers inocu- lated with culture 680-0 had a progressive increase in tuber- culin sensitivity. The mammalian tuberculin sensitivity was greater than the avian tuberculin sensitivity. The heifers inoculated with culture SOB-O had no sig- nificant increase in sensitivity to mammalian tuberculin. One heifer had a slight increase in sensitivity to avian tuberculin. Aerosol Experiment Nine calves, between 3 and 6 months of age, were divided into three groups of three calves each. Each group was inoculated with a different strain of bovine-origin Group—III mycobacteria by aerosol containing approximately 1.5 x 109 i 102 organisms. The calves were tuberculin tested as described previously. The calves were euthana- Lloyd Samuel Goyings tized and a necropsy performed ahead of the scheduled 2, 4 and 6 months after inoculation due to anticipated death. The six calves, three inoculated with culture SlC~O and three with culture 68C~O, had extensive lesions of pulmonary disease. The thoracic lymph nodes with afferent lymph vesn sels from the lungs also had extensive lesions. The micro- scopic lesions could not be distinguished from lesions found in cattle infected with Mycobacterium bovis. This was also true of lesions studied from the intrauterine experiment. The disease rapidly Spread to most of the lymph nodes, the abdominal organs (liver, spleen, kidney), and throughout the body, principally by the hematogenous route. The distribution of lesions in the six calves exposed by aerosol to cultures 510-0 and 680-0 was markedly similar to lesions found in naturally acquired bovine tuberculosis. Calves inoculated with culture 50B~O had no detectable gross or microscopic lesions. Group-III mycobacteria, indistinguishable from the strains inoculated, were recovered from most of the lymph node pools in which lesions were found. Acidafast organ- isms were recovered from the thoracic lymph nodes of the group of calves inoculated with culture SOB-O. Five of the Six calves, inoculated with culture SlC~O and 680-0, had a marked mammalian tuberculin sensitivity. Calves inoculated with SOB-O had a slight increase in sensitivity to avian tuberculin. MYCOBACTERIOSIS OF CATTLE INOCULATED WITH GROUP-III ORGANISMS OF BOVINE ORIGIN By Lloyd Samuel Goyings A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pathology 1965 ACKNOWLEDGEMENTS The author is grateful to, and wishes to express appreci- ation to, the following peeple for their help in making this thesis possible. To Dr. R. F. Langham for his guidance in stimulating me to do graduate work in pathology, his help in acquiring a postdoctoral fellowship for the first two years of my grad- uate program and, above all, his role in my pathology train- ing. To Dr. C. C. Morrill for my selection to participate as a pathologist on the tuberculosis project leading to these investigations and for his counsel and guidance of the research. To Dr. W. L. Mallmann for his counsel and guidance, his enthusiatic motivation in research and his significant part in making this research possible. To Dr. E. P. Reineke for his counsel and guidance in my graduate program, and particularly his philOSOphy as a scien- tific investigator. To Dr. S. D. Sleight for his fellowship, patience and helpful suggestions in the writing of this thesis. To. Dr. V. H. Mallmann for her assistance in preparation of the cultures; to Dr. J. A. Bay for conducting much of the tuberculin testing; and to both for their preliminary work leading to these investigations. ii To the technical staff of the tuberculosis project for their cooperation and assistance in performing the bacterio- logical and histOpathological procedures. To the United States Department of Agriculture through which this research was supported with contract-cooperative agreements. To Dr. R. M. Scott for his assistance in selection of the cattle for these investigations. To Dr. D. J. Ellis for his technical assistance in the intrauterine experiment. iii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . 1 REVIEW OF LITERATURE . . . . . . . . . . . . . . . . 4 MATERIALS AND METHODS . . . . . . . . . . . . . . . 23 A. Intrauterine Experiment 23 B. Aerosol Experiment 32 RESULTS. . . . . . . . . . . . . . . . . . . . . . . 43 A. Intrauterine Experiment 43 B. Aerosol Experiment 78 C. Tuberculin Sensitivity Studies 115 DISCUSSION . . . . . . . . . . . . . . . . . . . . . 128 A0 Intrauterine EXperiment 128 B. Aerosol Experiment 136 SUMMARY . . . . . . . . . . . . . . . . . . . . . . 143 A. Intrauterine Experiment 143 B. Aerosol Experiment 145 REFERENCES . . . . . . . . . . . . . . . . . . . . . 147 iv Table 1. IO. 11. 12. LIST OF TABLES Page Pagination of results according to animals and cultures used . . . . . . . . . . . . . . . 42 Summary of results of bacteriological exami— nation (acid-fast isolations) of lung tissue from guinea pigs exposed to aerosol with a culture of g. chlei . . . . . . . . . . . . . . 78 Tuberculin test results of inoculating heifers by intrauterine route with culture SleO . . . 116 Tuberculin test results of inoculating heifers by intrauterine route with culture cacao . . . 117 Tuberculin test results of inoculating heifers by intrauterine route with culture SOB=O . . . 118 Tuberculin test results of inoculating calves by aerosol with culture SlC~O . . . . . . . . . 119 Tuberculin test results of inoculating calves by aerosol with culture 680:0 . . . . . . . . . 120 Tuberculin test results of inoculating calves by aeI'OSOl With CUILUI’G SOB'O o o o o o o o o o 1.21. Location of macrosc0pic and microsccpic les- ions frcm intrauterine eXperiment . . . . . . . 122 Summary of bacteriologic results (acidwfast isolations) from heifers wLLn intrauterine inoculation . . . . . . . . . . . . . . . . . . 124 Location of macrosc0pic and microscopic lesw ions of calves inoculated by aerosol . . . . . 125 Summary of bacteriologic results (acidwfast isolations) of inoculating calves by aerosol . 127 Figure 1. LIST OF FIGURES Aerosol Apparatus A. Aerosol chamber B. Filter 0. Compressor D. Exhaust tube E. Inlet tube . . . . . . . . . . . . . Atomizer-Venturi Apparatus A. Primary air flow B. Secondary air flow C. Atomizer D. Venturi . . . . . . . . . . . . . . . Segment of uterus from heifer 73 62 days after inoculation with culture 6SC-O. Note raised nodules on mucosal surface . . . . . . Omentum from heifer 70, 124 days after inocu- lation with culture 68C~O. Note the multiple, raised, irregularly sha ed nodules in the serosal surface (arrows? . . . . . . . . . . . Omentum from heifer 70, 124 days after inocu- lation with 680-0. Note the area of necrosis, giant cells, surrounded by lymphoid and epi- thelioid cells. New Fuchsinc-H & E. x187 . . Uterus from heifer 71, 176 days after inocu- lation with culture 68C~O. Note the extensive tuberculous process with caseation (arrows) in the thickened walls . . . . . . . . . . . . Uterus from heifer 71, 176 days after inocuu lation with culture 680-0. Note the mucosal glands (arrow) filled with neutrOphils, epi- thelioid cells in the lamina pr0pria, and suppuration (l) in the area of the destroyed epithelium of the endometrium. New Fuchsinu- H & E. x187 . . . . . . . . . . . . . . . . . Another area of the uterus shown in Fig. 7. Note the epithelioid cells, several giant cells (arrows) mixed with leukocytes. New Fuchsin--H & E. x187 . . . . . . . . . . . . vi Page 34 34 56 61 61 67 67 71 Figure Page 9. Lung from calf 91, 44 days after aerosol exposure with culture SlC—O. There are sev- eral subpleural tuberculous lesions (arrows) . 81 10. Lung from calf 91, 44 days after aerosol exposure with culture 510-0. Note bron- chiole (arrows) filled with inflammatory exudate and surrounded by granulomatous process. New Fuchsin--H & E. x187 . . . . . 81 11. Higher magnification of the respiratory bronchiole shown in Fig. 10. Note the epithelioid cells, lymphocytes and a few neutrophils. New Fuchsin~~H & E. x400 . . . 83 12. Posterior mediastinal lymph node from calf 86, 76 days after exposure to culture 680-0. Note the size of the lymph node . . . . . . . 96 13. Lung from calf 83, 78 days after aerosol exposure with culture 680-0. Note extensive circumscribed foci and confluence of tuberu culous lesions . . . . . . . . . . . . . . . . 103 14. Posterior mediastinal lymph node from calf 83, 78 days after exposure to culture 680-0. The normal architecture was obliterated by necrotic tissue . . . . . . . . . . . . . . . 105 15. Posterior mediastinal lymph node from calf 83, 78 days after aerosol exposure with culture 680-0. Note area of caseous granu~ loma. New Fuchsin--H & E. x75 . . . . . . . 105 16. A different area of the same lymph node as in Fig. 15. Note the extensive cellular necrosis of the zone bordering the area of caseation necrosis. New Fuchsinn-H & E. x187 . . . . . . . . . . . . . . . . . . . . . 108 17. Higher magnification of zone of cellular necrosis shown in Fig. 16. There is ex- tensive pyknosis and karyorrhexis. New FuChSln--H & E. X400 . . . . . . . . . . . . 108 18. Kidney from calf 83, 78 days after aerosol exposure to culture 680-0. Note infarcted area (1) and several subcapsular tubercum lous lesions (arrows) . . . . . . . . . . . . 109 vii Figure 19. 20. 21. Kidney from calf 83, 78 days after aerosol exposure to culture 68C~0. area of caseation necrosis surrounded by a zone of H & E. granulomatous reaction. X75 0 o o o o o o o 0 New Fuchsin-- Kidney from calf 83, 78 days after aerosol exposure with culture 680-0. tensive tubular degeneration and calcifica- New Fuchsin--H & E. x75 tion. Higher magnification of tubules in Fig. Note the ex- 20. There is a marked coagulative necrosis and karyolytic loss of nuclei of the renal tubules. New Fuchsin--H & E. viii x187 There is a large Page 111 111 INTRODUCTION The decrease in numbers of tuberculin reactors in the cattle population in this country is rather impressive when one compares the incidence of the disease from 1917 to 1963 (5.0% and .10% respectively). This was accomplished mainly by the diligent and persistent use of the intradermal tuber- culin test as the diagnostic tool for removing tuberculin reactor animals from herds. Of greater significance, per- haps, from the public health standpoint has been the rapid decrease of animals with gross lesions. Only 1780 lesion reactors were reported in the United States Department of Agriculture (USDA) fiscal year 1961 (Wilder, 1962). However the eradication program is not without problems. During the last 20 years the decrease in the number of reactors was not significant when compared to the previous 20 Years. In fact there was an increase from 11 to 23 reactors of every 10,000 animals tested between 1952 and 1959 (Anon., 1960). This would suggest to the outside observer that the reaching of a plateau indicates in essence only a control program. Analysis of the post-mortem findings in reactors shows a marked increase in the percentage of "no gross lesion" (NGL) animals on necropsy. By 1961 (Wilder, 1962), the NGL animals represented approximately 73% of the reactors slaughtered. If the posi- tive tuberculin tests of the NGL animals are due to organisms other than pathogenic mycobacteria or to non-specific sub- stances, then the livestock industry is suffering an unneces- sary economic loss. This caused grave concern in the USDA which has the responsibility of conducting the tuberculosis eradication program. The entire eradication program needed reevaluation. A series of regional meetings was held begin- ning with a conference at Michigan State University in 1958 on tuberculosis eradication. There are many problems that must be resolved. Such problems include: 1) the need for a reliable test for diag- nosis; 2) the failure to demonstrate unequivocally the cause of the large percentage of NGL reactors; 3) the true relation- ship of the tuberculoid skin lesion to tuberculin sensitivity; and 4) the possible role that mycobacteria other than the classical organisms may have in causing tuberculin sensitivity (Hastings 2£.§l~’ 1930). A research program on bovine tuberculosis was set up at Michigan State University by contract grants from the USDA. The research was to be conducted by a team composed of staff from the Departments of Pathology and Microbiology and Public Health. Although there were scattered reports and brief mention of atypical mycobacteria in the human literature, no real emphasis was placed on these organisms until such reports as those of Timpe and Runyon (1954), Crow 23 §l° (1957) and the monumental work of Runyon (1959). More will be said about these reports later. It was soon recognized by different investigators (Mallmann gt al., 1961; Scammon 23 al., 1963; Yoder, 1964), that an analogous situation was present in the animal field. In our bacteriology laboratory, large numbers of acid-fast organisms were isolated from bovine tissues which appeared not to conform to the characteristics of classical mycobacteria. In order to evaluate the possible significance of these or- ganisms, laboratory animals, swine and cattle have been inoc- ulated primarily by the intradermal route. Some of the Group- III mycobacteria were found to cause disease and tuberculin hypersensitivity. Through a contract (No. 12-14-100-6852 (45) ) between the USDA and Michigan State University, investigations were conducted to determine the relative pathogenicity and signif- icance of different routes of inoculation. The research described herein is limited to studies of cattle inoculated by the aerosol and intrauterine routes with bovine-origin Runyon Group-III isolants of low and high virulence. Specific objectives included a study of the course of the disease in inoculated cattle by the use of tuberculin tests, serologic procedures and clinical observations. Fur- ther objectives included a detailed macroscopic, micros00pic and bacteriologic examination. REVIEW OF LITERATURE Isolation of mycobacteria which did not conform to the characteristics established by Koch in 1881 were soon recog» nized (Nocard and Roux, 1888) as reported by Xalabarder (1961). Scattered reports in the literature (Baldwin, 1942; Feldman, 1943; Karlson and Feldman, 1953; Buhler and Pollak, 1953) described clinical and bacteriologic findings, tuber~ culin sensitivity and animal pathogenicity studies of disease associated with atypical mycobacteria. In most laboratory situations the atypical mycobacteria were considered as sapro- phytes or contaminants, and were often discarded. This was understandable since there were many reports (Frey and Hagan, 1931; Karlson, 1958; Mallmann, 1963) of acid-fast isolations from innumerable inanimate materials. These organisms were considered ubiquitous and thus easily contaminated material submitted for culture. Not until sufficient clinical data had been accumulated and reported (Wood gt 31., 1956; Crow 33 al., 1957; Hall 33 31., 1957), did it seem necessary to reassess the possible significance of the isolation of atypical mycobacteria. The need for a working classification system for atypi- cal mycobacteria was emphasized by Karlson (1958) when he reported that by 1945 there were 163 known strains. Such a grouping was very ably devised by Runyon (1959). This clas- sification was based on human isolants collected from differ- ent sources throughout the United States. Preparation for this work was started by other investigators, particularly Dr. William Feldman who circulated detailed outlines on how to study these organisms (Runyon, 1959). The features used in classification included: 1) pretherapy drug resistance; 2) strong catalase activity; 3) capacity to grow at room temperature; 4) failure to produce progressive disease in guinea pigs; 5) colonies strongly pigmented or smooth, easily dispersed in liquid and with rapid growth; 6) niacin, peroxi- dase neotetrazolium reduction and microcolonial pattern in neutral red. The atypical mycobacteria were divided into four catem gories: Group I (photochromogens); Group II (scotochromo- gens); Group III (nonphotochromogens); and Group IV (rapid growers). The Group-III strains had features which over~ lapped with strains of M. avium, M. tuberculosis and M. bovis. Probably the most distinctly different feature was their pathogenicity for laboratory animals. Human isolants clas- sified as belonging to Group III were not uniform in their pathogenicity. Some strains were found to be no more patho- genic than Group I (Runyon, 1959) which did not produce pro- gressive disease when inoculated subcutaneously with 5 mg. (dry weight) of the organisms. The author further stated that when 1 mg. was inoculated intracardially, death often resulted in 4 to 5 weeks. When 0.01 mg. was inoculated intravenously or 3 mg. intraperitoneally, disease was mani— fested by formation of lesions in lungs, liver, spleen and kidneys. Other Group-III strains produced no detectable lesions. Later, Corpe gt 21° (1963) reported that as a result of the lack of pathogenicity for the recognized laboratory ani- mals used in tuberculosis diagnostic laboratories, there was no standard test for mycobacterial pathogenicity. This was considered unfortunate since the relative significance of an isolant depended on the closeness and consistency of association of the recovered organisms with the clinical and pathologic findings of the disease since it was impossible to consistently fulfill Koch's postulates. The problem was compounded by the many isolations of acid-fast mycobacteria from apparently healthy individuals (Edwards 33 al., 1959; Atwell and Pratt, 1960). Other workers investigated the pathogenicity of atypical mycobacteria for laboratory animals either by using different routes of inoculation or different laboratory animals in an attempt to find a suitable way to evaluate virulence. Pollak and Buhler (1955) described the pathogenicity of the so» called yellow bacillus (classified in Group I by Runyon, 1959) for the guinea pig, hamster, rat, mouse, rabbit and chicken. The organism did not produce progressive disease in guinea pigs although there was a lesion at the site of inoculation and microscopic lesions in some of the visceral organs 2 to 4 weeks after inoculation. Intramuscular and intraperitoneal inoculation failed to produce either gross or microsc0pic lesions in chicks. Likewise no disease was noted in the white rat. The authors concluded that the ham- ster was the animal of choice, since consistently progressive disease was produced when large numbers of these organisms were inoculated intraperitoneally. Although mice were gen- erally susceptible to the organisms, there appeared to be considerable variability in the results. Similar results were reported by Feldman (1963) using Group-III mycobacteria. He inoculated hamsters in the substance of each testis (0.001 mg.) and under the dermis of the concha of each ear (0.005 mg.). Under the conditions of the experiment, irreversible pathologic changes occurred in the hamsters after intratesticular inoculation. Chickens and rabbits were also inoculated with the same mycobacteria. Although the rabbit was resistant, a formidable disease occurm red in chickens when infection was induced by intracerebral or by intravenous inoculation. Kite e; §l° (1952) used the intradermal and subcutaneouS* routes of inoculation in guinea pigs in an attempt to dist tinguish between saprophytic and virulent mycobacteria. A comparison between the two routes of inoculation was made with 266 unknown cultures. The guinea pig was inoculated into the right groin with 1 ml. (0.1 mg) and 0.1 ml. (0.01 mg.) intracutanecusly in the axillary or inguinal region of the abdominal wall, with the same suspension. There was a close correlation between the subcutaneous and intracutaneous routes of inoculation. A virulent organism produced an ul- cer in 2 to 3 weeks. The saprophytic organism occasionally produced a nodule which seldom ulcerated and usually healed within six weeks. The test was considered positive if there was an ulcer at the site of inoculation and if acid-fast organisms could be demonstrated in the regional lymph node. Kubica gtpgl. (1960), using the same technique, studied the comparative virulence of atypical mycobacteria and clas- sical mycobacteria. Guinea pigs were inoculated with 0.1 mg., 0.01 mg. and 0.001 mg. intracutanecusly in the shaved abdominal skin. Of the atypical mycobacteria, Group-I and Group-III strains were the most consistent in producing ulceration. Approximately 50% of the scotochromes (Group II) and 30-40% of the rapid growers (Group IV) produced ulcera- tion at the site of inoculation. The author concluded that the intracutaneous route of inoculation appeared to give a better measure of the virulence for man than the subcutan~ eous, intraperitoneal or intracardial route. No data were given on the condition of the lymph node draining the site of inoculation. One report (Durr 33 al., 1959) described the use of saprophytes (M. pglgl and M. smegmatis) as a control when studying the extent of pathology produced by the atypical and classical mycobacteria. Three-to-six-week-old chickens were inoculated subcutaneously (Experiment I) or intravenous- ly (Experiment II) with 1.0 ml. and 0.4 m1. of diluted culture, respectively; adult golden hamsters were inoculated intraperitoneally with 1.0 ml. of diluted culture, guinea pigs were inoculated subcutaneously with 1.0 ml. of diluted culture and mice were inoculated intravenously with 1.0 m1. of diluted culture. Eight weeks after inoculation the aniu mals were euthanatized. The results are summarized in the following table: Guinea Chicken gig Hamster Mggse M. tuberculosis __ +++ +++ + M. 3112M ++ __ + + Nonphotochromogens i __ i __ Photochromogens i __ ++ ++ SaprOphytes male; _ _ _' _ M. smegmatis Progressive disease resulting in death of all of the animals in a particular group is indicated by +++; extensive gross disease without a fatal outcome by ++; microscopic disease only by +; and microscopic disease in some, but not all, of the animals in a particular group, by 1° The disease caused by atypical mycobacteria in man com: monly takes the form of a chronic systemic disease involving principally the lung but sometimes also lymph nodes, skin, meninges and joints (Wood §§,al., 1956; Crow 21 a;., 1957; Weed 23 al., 1956; Merckx 33 §;., 1963). Occasionally, the disease was generalized (Wood 23 31., 1956; McCusker and 10 Green, 1962). The lesions were indistinguishable from lesm ions caused by classical tubercle bacilli as was well exem- plified by Corpe and Stergus (1963) who submitted a group of representative tissue sections from Group-III and M. EERQE- culosis infected patients to 27 pathologists who had an interest in tuberculosis. Of this group, 53% did not distinguish between the causative organisms by studying the histopathologic material. This may imply that 47% could distinguish between the organisms from studying the histo- pathologic changes but, statistically, similar results could be obtained by merely tossing a coin (Dixon and Massey, 1957). The disease was found more often in white males in the 3rd, 4th, 5th and 6th decades of life (Runyon, 1959; Merckx 25 gl., 1963). There was little or no concrete evidence of transmission from one person to another (Merckx e3 gl., 1963). In a rather high percentage of cases, there were compli- cating or contributing factors such as emphysema, anthra- cosis, silicosis, bronchiectasis, etc. (Runyon, 1959). Although the epidemiologic data were limited, there appeared to be a definite geographic distribution of the different groups of atypical mycobacteria (Merckx 33 gl., 1963; Edwards g; a;., 1960). There is ample evidence that the presence of atypical mycobacteria is not a new phenomenon in the animal kingdom. As early as 1930, Hastings 23.2l° reported recovery of acid_ fast bacteria from "no gross lesion" reactors. This was followed by a report by Hagan (1931), who isolated acid-fast 11 mycobacteria from mesenteric lymph nodes with no detectable gross and microsc0pic lesions. The isolants were considered to be saprophytes from the intestine which could cause tuber- culin sensitivity. Feldman (1938) described the isolation of M. aylMM-like mycobacteria from swine and chickens which did not manifest the usual pathogenicity for chickens. The organisms proba- bly would be classified as nonchromogenic acidufast bacilli under the current grouping of atypical mycobacteria. In another report (Karlson and Feldman, 1940) nonchro~ mogenic acidufast microorganisms were recovered from 25% of 94 swine tonsils. The organisms failed to produce detectable disease in chickens, mice and calves. Also, little if any virulence was noted when they were inoculated into guinea pigs and rabbits. However, the organisms induced sensitiv- ity to avian tuberculin and homologous culture filtrates, but not to mammalian tuberculin. Unidentified mycobacteria were cultured from the retro: pharyngeal and ileocecal lymph nodes and spleens of 100 apparently healthy cattle (Smith, 1954, 1958). An acid—fast bacterium identified as Mycobacterium lacticola was isolated from the milk and mammary tissue of cows with tuberculoid mastitis (Stuart and Harvey, 1951). The condition was only in those animals infused for mastitis with an oily vehicle. The affected animals did not react to either avian or mammalian tuberculin. Another report (Tucker, 1953) described the isolation of mycobacteria from 12 a herd which had an incidence of 21.7% infection. Previous to examination, there were 14 cows which reacted to the tuberculin test. The organism appeared to be identical to Mycobacterium fortuitum. The disease was characterized by multiple granulomas. The udders of these animals had also been infused with an oily suspension for mastitis treatment. Nonchromogenic acid-fast bacilli were isolated from tuberculous swine (Scammon g3 gl., 1963). Of 63 cultures studied, 43 strains were swine isolants, 10 were avian strains and 10 were Battey strains. Forty of the 43 strains of swine origin consistently produced death in chickens while all of the avian strains produced typical avian tuber- culosis. The Battey strains did not produce death at three months in chickens. The author concluded that none of the strains could be distinguished from each other using the established criteria for GroupnIII organisms. Approximately 100 strains of nonchromogenic acidwfast bacilli were isolated from a university swine herd (Mallmann _£ _;., 1962). Isolations were made from swine which had gross lesions and from those with no gross lesions. This prompted a bacteriologic survey to determine the geographic distribution of these organisms. Tissues with lesions were collected from abattoirs processing swine from Illinois, Missouri, Ohio, Indiana, and Michigan. Eighty-five percent of the isolants were M. gyigg and the remainder were Group- III mycobacteria. The Group-III strains were inoculated intradermally into swine and guinea pigs and intraperitoneally 13 into chickens. No lesions were demonstrated in chickens although the organisms could be recovered. There was a skin lesion characterized by ulceration at the site of inocula- tion in all the swine and guinea pigs inoculated. At necropsy, lesions were found in some of the swine, involving lymph nodes of the head and the lymph node that drained the site of inoculation. No gross lesions were detected in the guinea pigs at necropsy. Another report (Mallmann 33 gl., 1964) described the bacteriologic, allergenic, and pathogenic investigations from a sample of 40 out of 300 isolants. These organisms were recovered from bovine and swine tissues. The organisms appeared to be highly heterogeneous and variable in their characteristics. Some resembled the isolants made from man and classified into Runyon's grouping. A few of the strains appeared to be between Group I and II and were called pseudo- chromes. With some strains, virulence was increased follow- ing reisolation and reinoculation. The intradermal route of inoculation was used to give presumptive evidence of viru- lence. A skin lesion at the site of inoculation confirmed the virulence of the organism. McGavin (1964) and McGavin g; 3;. (1964) found that when Group-III mycobacteria of bovine origin were inoculated intradermally into calves, there was considerable variability in pathogenicity of the organisms. 0f 14 calves, using 7 cultures for inoculation, six calves had generalized disease, three had lesions at the site of inoculation and the lymph 14 node draining the inoculation sites (primary complex), three had lesions at the site of inoculation only, and two had no detected gross or microscopic lesions. There was consider- able variability in the extent of the disease produced with the same culture. Of 10 of the 14 calves on which a compar- ative cervical tuberculin test was conducted; the three cases with generalized disease had positive tuberculin tests in the caudal fold and positive mammalian and avian tuberculin tests in the cervical region; the two cases with lesions sufficient to be called a primary complex had conflicting results. One calf had a positive tuberculin caudal fold test but a negative comparative test, although the avian response was 10 mm. larger than the mammalian response. The other calf had only a 1.5 mm. increase at the caudal fold. The remaining five calves were considered negative. It therefore appeared that the Group-III mycobacteria caused tuberculin sensitivity and sensitivity was dependent on the extent of disease occurring in the inoculated animal. McGavin (1964) and McGavin gt §l° (1964) also found that Group-III isolants of swine origin had little pathogenicity for calves. Seven cultures were inoculated intradermally into calves. Only one culture produced significant lesions, which consisted of a caseo-calcareous granuloma in the lymph node draining the inoculation site and an abscess at the site of inoculation. The calf inoculated with this culture had a negative caudal fold test with mammalian tuberculin and was negative to avian tuberculin in the cervical region. 15 The Group-III mycobacteria of feed and soil origin and pseu- dochromes of bovine origin were not pathogenic for calves. Likewise the Group-IV mycobacteria of bovine origin produced no significant lesions in calves, except for one culture in one calf. This animal had lesions at the site of inocula- tion and the draining lymph node. Inasmuch as 10 mg. (wet weight) of culture was used and the animal was stressed by an unintentional exposure to disinfectant aerosol, little significance was attached to this finding. It was not pos- sible to differentiate between lesions produced by M. 32113 and by atypical mycobacteria. The calves inoculated with Group-III organisms of feed and soil origin, pseudochromes of bovine origin and Group-IV organisms produced little or no sensitivity. Two calves, one inoculated with a pseudo- chrome and the other with a Group-IV mycobacterium, had a caudal fold response of 6 mm. and 7 mm. respectively. Both of these animals were negative to the cervical test. Cattle have been sensitized to tuberculin by mycobac- teria other than M. 33113 and atypical mycobacteria (McGavin, 1964). These included M. paratuberculosis, M. tuberculosis, and M. 3113M (Feldman, 1960; Johnson 31 31., 1961; Wilder, 1962). A significant problem related to tuberculin sensitivity is the so-called "skin tuberculosis" of cattle. The first report was by Traum (1916), who described the clinical con- dition as a granulomatous process associated with acid-fast bacilli which could not be grown and identified. Hole and 16 Rules (1939) grouped the lesions on a pathologic basis. Type-I lesions were cellular and had many giant cells and little necrosis; type II had multiple areas of necrosis and calcification; and type III had encapsulated foci of lique- faction which ulcerated and discharged pus. The lesions varied similarly in material studied by Daines and Austin (1932), Hedstrom (1949) and in this laboratory (Goyings, 1962). Hedstrom (1949) studied 606 specimens and found that 20% could be called type III, 73% type II and the remainder type I. In this laboratory (Mallmann, 1962; Goyings, 1962), out of 351 cases of "skin lesions" studied, acid-fast organ- isms were isolated from 42% of the cases. Two isolants were classified as Group I, 14 as Group II, 39 as Group III, 17 as pseudochromes, 34 as Group IV, and 42 were not classified. Most of the lesions were type II or III and at least some of the types I and II had undergone ulceration. Differences in the type of lesion appeared to be due to the stage of the disease and not to the mycobacteria isolated. The relation- ship of the isolation of acid-fast bacteria to the spontan- eous lesions was not known. Other workers (Beach and Hastings, 1924; Hastings 31 31., 1924; Carpenter and Goldberg, 1925; Mitchell, 1928; Day, 1928) had been either unsuccessful in culturing the acid- fast organisms or unsuccessful in reproducing the disease. Although acid-fast organisms were isolated from skin lesions as a result of the extensive work of Daines (1938), 17 the production of sensitivity and lesions in cattle was not constant and the lesions were only examined clinically. The only apparent reproduction of the condition was by Hedstrom (1949) who was able to produce typical lesions and tuberculin sensitivity in 3 of 25 cattle inoculated using a composite sample of 4 spontaneous lesions. Of particular interest is the fact that the condition usually remains localized. Runnells (1932) did not find involvement of the regional lymph nodes in a series of skin- lesion cases studied. However, Krantz (1938) reported involve- ment of the regional lymph node in 12 of 32 cases. Others (Thomann, 1949; Role and Hulse, 1939) have observed lesions in some regional lymph nodes. The lesions were generally seen only on histOpathologic examination and were character- ized by a granulomatous inflammatory process. Acid-fast bacilli were demonstrated in some of the microscopic lesions. Willigan (1961) studied histopathologioally tissues from 56 tuberculin reactors and found 38 (67.9%) with microscopic granulomas in the carcass lymph nodes (primarily the right and left prescapular but occasionally the right and left prefemoral lymph nodes). A later review (Goyings, 1962) revealed that, out of 179 tuberculin reactors in which carcass lymph nodes were studied and in which skin lesions were not found or submit- ted, micros00pic granulomas were found in 79 (44%). Gross lesions were found in 3% of the carcass lymph nodes. Skin lesions, organs and lymph nodes from 35 tuberculin reactors 18 were studied histopathologically. Twenty-one (60%) of the carcass lymph node pools (one pool per animal) had micro— scopic granulomas. The lesions were characterized by few to numerous scattered foci of epithelioid cells in the cor- tices of the affected lymph nodes. In many cases giant cells were a component of the inflammatory reaction. Casea- tion and calcification were not Salient features. Three reactors (9%) had gross lesions, acid-fast organisms were found in 7 (20%) and acid-fast organisms were isolated from 2 (6%). According to Paterson (1956), much of the data con- cerning the skin lesion problem as a cause of tuberculin sensitivity has been biased, since a search for the lesions has been made only when anomalous tuberculin reactions were found. There are very few data on skin lesions in tuber- culin-negative herds. The problem of mycobacterial classification was diffi- cult if not almost impossible due to the complexity of the organism (Xalabarder, 1961). The use of bacteriologic tech- niques for primary isolation was considered essential by Karlson (1964) with better laboratory techniques and the recognition of strains of M. tuberculosis less virulent for guinea pigs and of atypical mycobacteria as potential patho- gens. Yet in another laboratory (Ekdahl and Macfarlane, 1963) it was pointed out that potential pathogens could be overlooked since primary isolation of mycobacteria by guinea l9 pig inoculation was considered more sensitive than bacterio- logic techniques. Feldman (Hull, 1963) considered bacteriologic findings to be subject to considerable variation and thus only suf- ficient for presumptive identification; therefore laboratory animals (guinea pigs, rabbits, chickens) were needed for final identification of M. 33313, M. 31133 and M. tuberculom 313. Inasmuch as the atypical mycobacteria did not cause disease in guinea pigs, bacteriologic findings formed the basis of identification by Runyon (1959). Karlson (1958) questioned the use of "atypical" or "anonymous" and stated that the 6th edition of Bergey‘s Manual (Breed 33 31., 1948) listed them as Mycobacterium 333. However, subsequent editions have not used this desig- nation. Examples of conflicting mycobacterial identifica- tion illustrating this confusion can be found in many literature reports. Karlson 33 31. (1962) described a yellow-pigmented strain of Mycobacterium avium isolated from tuberculous lesions in a trumpeter swan. The organism was classified by one laboratory as a skotochromogen even though virulence for chickens and guinea pigs was present. In another report, the same author in 1964 stated: "....certain 13 vitro features characterize M. bovis and may lead to a presumptive identifi- catIon. The identification of M. bovis must be confirmed by animal pathogenicity tests." 20 In still another laboratory (Kubica, 1964) animal inoculations were avoided whenever possible due to their supposed unreliability. From our laboratory, a culture was submitted to two different laboratories for identification (Mallmann, 1964). The culture had been typed as a Group III. Laboratory 1 identified the culture as a Group III and Laboratory 2 as M. bovis. This problem was ably reviewed by Xalabarder (1961) who stated that: "....there is only one thing about them (mycobacteria) that is absolutely typical, and that is their great plasticity and adaptability to an extreme variety of environments. To suit those conditions, these organisms will change not only their metabolic patterns and morphologic characteristics, but even their mode of repro- duotion........a1though there are a great many exceptions to this rule, the presence or absence of pigment has been one of the factors which has been used by Penso and associates (1949) and more recently by Runyon (1959) to classify myco- bacteria. And yet, if the past history of bac- teriology is studied, it must be noted what little value can be attributed to pigmentation as a reliable criterion for differentiation." Even the relative pathogenicity of the saphrOphytic and pathogenic strains has been considered unreliable. Xalabarder (1961) found that when guinea pigs were inocu- lated with saprophytic strains and kept for a much longer period than two months, histopathologic lesions developed which were characterized by epithelioid and giant cells with marked fibroblastic proliferation. Reinoculation into 21 guinea pigs caused caseation of the mediastinal lymph nodes and fibroblastic proliferation of all organs. The cytochemioal reactions appeared to have limited value in classification. Hauduroy (1955) found that 50% of the strains tested showed discrepancies. Possibly this problem could be at least partially alleviated if quantita- tive rather than qualitative tests were used. This was emphasized by Wayne (1964) who stated that: "....time complicates the problem of applying biochemical tests to classification of mycobacteria by virtue of the great range of rates of their meta- bolic reactions. One must take into account inocu- lum and substrate concentration, time of observation, and magnitude of reaction." The trend to apply computer methods for bacterial classification was discussed by Floodgate (1962). His approach was based on comparison of reaction kinetics on a large group of organisms with a high agreement with one another. The author concluded that selection of characters for classification were based on natural frequency distribu- tions of randomly selected organisms. Further computer analysis avoided the arbitrary selection of characters and thus reduced the bias that was introduced into a classifica~ tion. The raw data dictated the boundaries of a grouping. According to Wayne (1964), the development of an accept- able and useful taxonomic mycobacterial classification will await a group of research workers representing a number of disciplines with sufficient scope and competence to study the problem with computer methods. The author concluded 22 that the group of mycobacteria selected for study should be taken from the natural pOpulation of mycobacteria on a ran- dom basis. MATERIALS AND METHODS A. Intrauterine Experiment 1. Source of Animals Nine heifers were obtained by Dr. R. M. Scott, Animal Disease Eradication Division (ADED), USDA, Lansing, Michigan from a Mississippi herd of dairy cattle. These animals had a history of no tuberculosis reactors and were essentially negative to the caudal fold test using 0.1 ml. mammalian tuberculin. They were also negative to a cervical test using avian tuberculin, mammalian tuberculin and Johnin. The heifers were Holstein, Guernsey and Holstein-Guernsey crossbreds. The animals were infected at approximately 21 to 23 months of age. 2. Isolation Procedures The heifers were divided into three groups and so kept, 3 animals to each isolation room, until necrOpsy. Separate boots, rubber aprons, jackets and disposable gloves were provided for personnel entering each room. In addition, the attire which was worn but not changed between rooms included disposable cap and mask and a pair of coveralls. Each time the personnel left the room, the boots, apron, Jacket and any instruments removed were washed in disinfectant? *Torsite, The Dow Chemical Company, Midland, Michigan. 23 24 The heifers were fed a balanced ration consisting of coarsely ground alfalfa hay mixed with a mineral-supplemented concentrate. Water was supplied ad libitum. Since no bedding was used on the floor, the feces were disposed of in the sewage system. 3. Inoculation of the Animals Each group of 3 heifers was inoculated with a different strain of Mycobacterium. The strains were Runyon Group 111's of bovine origin, selected on the basis of previous pathogen— icity and sensitivity studies in laboratory animals and calves (McGavin, 1964). One strain, culture BOB-O appeared to be of low viru- lence and had greater sensitivity to PPD-Bf The other two strains were of higher virulence, culture 510-0 and culture 68C-O. The animals were injected with pregnant mare serum (PMs)** at a dosage of 20 ml.(l0,000 units) subcutaneously, 2 days prior to inoculation, in an attempt to produce estrus. Bull semen*** diluted with buffered semen**** extender, but without antibiotics, was used to inseminate the heifers. *Purified Protein Derivative-Battey strain (produced by Seibert's method), received from L. Edwards, Communicable Disease Center, Atlanta, Georgia. **"Gonadin", Ashe Lockhart, Division of Haver-Lockhart Laboratories, Shawnee, Kansas. ***Obtained through courtesy of Michigan Animal Breeder‘s Cooperative, East Lansing, Michigan. ****Cornell University Extender (egg-yolk-citrate extender), courtesy of Michigan Animal Breeder s COOperative, East Lansing, Michigan. 25 One mg. (wet weight) of mycobacteria (approximately 1 x 108 organisms) was placed in a disposable insemination tube for each of the nine heifers. The inoculum was sealed in the tube by introducing a plug of vaseline on each side of the culture. The insemination tube was introduced by rectal manipula- tion into the body of the uterus through the external 08 of the cervix. A disposable lO-ml. syringe filled with 5 ml. of extended bovine semen was attached to the insemination tube. With gentle pressure the inoculum and the semen were forced into the uterus. The insemination tube and syringe were disposed of by incineration. 4. Clinical Examinations The heifers were observed weekly for the first six weeks and semiweekly thereafter for signs of disease. Ob- servations included rectal temperature, condition of the animal and rectal examination. Blood samples (8, 20—ml. tubes per heifer) were taken every 2 weeks throughout the experiment for serologic examination (hemagglutination test, etc.). 5. Tuberculin-Test Technique All of the heifers were tuberculin-tested one week prior to each scheduled necropsy or one week prior to 2, 4 and 6 months after inoculation. The left caudal fold and the left cervical neck area were used for the sites of injec- tion. After clipping the hair, the skin thickness at the 26 site of injection was measured before injecting tuberculin (zero hour reading) using a Hauptner dermal thickness gauge* (used for all tuberculin measurements). The left anterior proximal third of the neck was in- jected intradermally with 0.1 ml. of avian tuberculin**; the left middle third of the neck with 0.1 ml. of mammal- ian tuberculin***; and the left posterior third with 0.2 ml. of johnin****. The left caudal fold was injected intradermally with 0.1 ml. of mammalian tuberculin. The skin thickness at the site of injection was measured at 24, 48 and 72 hours after injection of tuber- culin. All measurements were recorded in millimeters. The comparative test results (subtraction of corrected mammalian tuberculin response from corrected avian tuberculin response) was also recorded. The interpretation of the tuberculin tests was based on recommendations of other workers (Boddie, 1962; Anon., 1962). *H. Hauptner Co., Factory for Vet. Instruments, 565 Salingen, Germany. **Tuberculin, avian, intradermic, produced for the Agricultural Research Service (ARS), USDA. ***Tuberculin, mammalian, intradermic, produced for the ARS, USDA. ****Johnin, intradermic, produced for the ARS, USDA. 27 6. Necropsy Technique The necrOpsy technique was essentially based on that of Jones and Gleiser (1954) and modified by McGavin (1964) for the existing facilities. Prior to euthanatizing the animal, approximately 32 (20-ml.) tubes of blood were removed from the jugular vein for serologic procedures. The animal was given sufficient chloral hydrate (approximately 15 ml. of 40% solution per 100 lbs. of body weight) to cause anesthesia. The animal was placed on its left side and the right foreleg was dis- sected free from the thoracic chest wall and reflected back. The right common carotid artery was then exposed and incised. Euthanasia was performed by rapid exsanguination. Identification tags were removed and saved for future reference. The right foreleg was dissected free from the animal and the right prescapular and axillary lymph nodes removed. All lymph nodes removed from the carcass were either individually labeled and placed in individual plastic sacks or pooled and placed in plastic sacks for transporta- tion to the bacteriological laboratory. The lymph nodes and organs saved for bacteriologic examination were kept separate from those lymph nodes and organs saved only for histopathologic examination. At all times, attention was given to careful dissection of the lymph nodes from their respective locations to prevent internal contamination. 28 The right hindleg was dissected from the carcass and the right prefemoral, p0pliteal, ischiatic and left and right superficial inguinal lymph nodes were removed. The attendant then made a ventral midline incision through the skin and, after dissecting it free from the abdominal and thoracic cavity reflected it laterally. Then, the abdominal cavity was opened by cutting from the dorsal costal arch to the xiphoid cartilage, finally posterior along the linea alba to the pubis. The abdominal wall was reflected dorsally. During this time a ventral midline incision was made from the symphysis mandible to the cariniform cartilage of the sternum. The tongue was dissected from the mandible and the submaxillary lymph nodes removed. The soft palate was cut transversely and the greater cornua of the hyoid bones dissected from the larynx. The tongue, esophagus, larynx and trachea were then dissected from their attach- ment posteriorly to the thoracic inlet. The right and left medial and lateral retropharyngeal lymph nodes were identi- fied and dissected free. The right and left parotid lymph nodes were similarly removed. With the help of the attendant, the costal attachments of the diaphragm were dissected free and, with the use of rib shears, the dorsal ends of the ribs were cut and, simi- larly, the distal ends. The right chest wall was removed. Being careful not to contaminate the organs, a segment of the right diaphragmatic lobe of the lung was removed for culture. .219 Generous pieces of liver and spleen were also removed for culture. The attendant removed the intestine and dis» sected the mesenteric and colic lymph nodes free from the mesentery, as described by Jones and Gleiser (1954). The neck organs and lungs were removed in 2333. The right and left bronchial lymph nodes, right apical bronchus, anterior and posterior mediastinal lymph nodes were removed and placed in individual plastic bags. The esophagus and trachea were opened; the lung palpated and cut tranversely at intervals for lesion location and identification. The forestomachs were removed and their respective lymph nodes removed and pooled. While the attendant disarm ticulated the head and removed the skin; the right and left deep inguinal, left and right internal iliac and lumbar (including renal) nodes were removed and pooled separately. The liver was removed by cutting the diaphragmatic attachw ment and the hepatic lymph nodes removed. The liver was incised transversely approximately every cm. for lesion identification. The uterus, cervix and ovaries were removed in lggg by ligation of the vagina at the posterior external os cf the cervix. The organ was placed in a plastic bag for bacteriological examination. The kidneys were removed and cut transversely at intervals. Representative tissue was taken from the heart, kidney intestine, liver and spleen and fixed in 10% buffered formalin and Zenker"s fluid. A sample of lung was fixed in 30 formalin. Approximately six inches of ileum were tied off and removed for bacteriological examination. The brain was removed and representative tissue samples, as described by McGavin 33 El- (1962), were fixed in 10% buffered formalin for histOpathologic examination. The carcass was turned onto the right side. The left foreleg and hindleg were removed and the following lymph nodes removed and placed in individual plastic bags: left prescapular, axillary, prefemoral, popliteal and ischiatic. The mammary gland was cut transversely at intervals of about 1.5 cm. and, if lesions were detected, samples were saved for histopathologic examination. 7. Bacteriologic Technique All lymph nodes and organs removed from the carcass were taken to the bacteriological laboratory to be examined under an isolation hood. The lymph nodes and organs for bacterio- logic examination were trimmed of excess adventitial tissue and washed 5 times for 5 minutes each in 5% sodium hypo- chlorite solution. The lymph nodes were sliced every 2 to 3 mm. and examined for lesions. The organs were incised and a representative sample taken for bacteriologic examination. Sterile instruments were used for culturing each pool. Depending on the size of each pool, they were either blended in a Waring blendor with nutrient broth or ground with nutrient broth in a mortar with pestle. An equal amount of NaOH (4%) was added to the tissue homogenate. The sample was shaken and allowed to stand for 15 minutes and the pH adjusted to 7 by adding sufficient HCL (2%). The sample was then centrifugalized and the supernatant removed and seeded on LowensteinaJensen medium, Dubos Oleic agar and Middlebrook 7HlO agar. The tissue pools examined bacteriologically included the following: A. Right and left submaxillary, right and left medial and lateral retropharyngeal, and right and left parotid lymph nodes. B. Right and left bronchial (including the right apical bronchial lymph node), and anterior and posterior mediastinal lymph nodes. C. Mesenteric, colic, hepatic, and forestomach lymph nodes. E. Lung. G. Right and left superficial inguinal (supramammary) lymph nodes. H. Intestine. I. Liver and spleen. J. Right and left internal iliac and right and left deep inguinal lymph nodes. K. Uterus. 8. Histopathologic Technique Representative tissue was fixed in 10% buffered formalin from each lymph node and organ mentioned above except those 32 in which tissues had been saved at the time of necropsy. In addition to the above, the following lymph nodes were incised and representative tissue saved for histopathologic examina- tion: right and left prescapular, right and left axillary, right and left prefemoral, right and left ischiatic and right and left popliteal lymph nodes and omentum. The tissues were embedded in a tissue embedding medium* and cut at 6 microns thickness. The sections were stained with new fuchsin-hematoxylin and eosin (Willigan, Garric and Trosko, 1961). The following criteria were used to evaluate the tissues: 1) presence and description of gross or micro- scopic lesions; 2) lesions at the site of inoculation and regional lymph nodes; 3) generalized lesions either by lymphogenous or hematogenous route; 4) extent and distribu- tion of the lesions; 5) progressive or nonprogressive (Feld- man, 1943). B. Aerosol Experiment 1. Source of Animals Nine purebred Jersey male calves were obtained by Dr. R. M. Scott, ADED, USDA, Lansing, Michigan, from a Mich- igan herd. The herd had a history of being negative to the caudal fold test using 0.1 ml. of mammalian tuberculin. The calves were negative to comparative cervical tests using *Paraplast, Arthur H. Thomas 00., Philadelphia, Pennsylvania. 33 0.1 ml. of avian tuberculin, 0.1 ml. of mammalian tuberculin and 0.2 ml. johnin. The calves varied in age from 3 to 6 months at the time of inoculation. Approximately two weeks after arrival, the calves were castrated by clamping the spermatic cord with Burdizzo forceps. 2. Isolation Procedures The calves were divided into 3 groups, 3 animals to an isolation room. Strict isolation procedures as outlined in the intrauterine experiment were followed. 3. Aerosol Apparatus A rectangular-shaped box large enough to house the animal was used for the experiment (Fig. l). The box had previously been used for experimental euthanasia and thus contained some of the safety requirements needed for the experiment and required little modification. An inlet 2 inches in outside diameter (O.D.) was placed in the center of the door. This was connected to a tee 1 inch in inside diameter (I.D.). Two extension pipes, 16 inches long (1 inch I.D.) and each drilled with 5 5/8 inch holes, were attached to the tee running vertical to the box. This insured uniform dispersion of the aerosol cloud within the box. An exhaust outlet (1 inch O.D.) was placed in the center of the end of of the box. The inside measurements of the box were 29.5 inches wide by 46.5 inches high by 58 inches long, with a total of 46.04 cubic feet or 1303.67 liters of air displace» ment. Fig. 2. UQUJP Aero 34 . >"“ “-Rfifliigm 7,. ,_ ‘3‘ 163 i: sol Apparatus Aerosol chamber Filter ‘ Compressor Exhaust tube Inlet tube Atomizer-Venturi Apparatus Primary air flow Secondary air flow Atomizer Venturi 35 The external outlet was connected to a filter (Waxler, 1961) (using PG 50 glass-wool mat*) by tygon tubing (1 inch I.D.) and from the filter to the inlet of a two-cylinder compressor. A 25-ft. hose (3/4-inch I.D.) was connected to the outlet of the compressor and to a 25~gal. can of ortho- phenylphenol disinfectant-detergent** (0.5%) to destroy any organisms that might pass through the filter. The hose out» let was placed at the bottom of the can and covered with screening. This caused a breakup of large bubbles and in- sured better contact of any organism with the disinfectant. An atomizer-venturi apparatus (Wells 33 31., 1948) was constructed from glass and mounted on the tOp of the aerosol box (Fig. 2). This was connected to the inlet on the aero» sol box by 6 feet of tygon tubing (2oinch I.D.). The atomu izer was calibrated using culture media and 20 lbs. of air pressure. It was found that an average of 15 ml. of culture medium could be atomized per hour (.25 ml./min.). This air will be referred to as the primary air flow. The secondary air movement which consisted of air pas- sed through the venturi apparatus with the compressor in Operation was calibrated. The volume Averaged approximate- ly 21.8 L./min. If a culture with a cell count of 108“9 per ml. were used, and loss of viability and change in primary air flow were negligible, there would be approximately 1.14 *American Air Filter Company, Louisville, Kentucky. **Torsite, The Dow Chemical Company, Midland, Michigan. 36 x 107 i 102 organisms/L. air/min. flow through the aerosol chamber. The organisms were suspended in 0.1% bovine albumin and 0.1% Tween 80. This suspension maintains stability of the organisms in the aerosol cloud (Middlebrook, 1952). Tests were made to determine the effectiveness of the aerosol cloud, and its potential ability to infect lung parenchyma of guinea pigs. The aerosol cloud contained particles less than 5 microns in diameter as demonstrated by an Anderson Air Sampler (Anderson, 1958). Two tests were made with guinea pigs. The guinea pigs (5 each) were caged in a small wire cage and placed in approximately the same position. The animalsI external nares were held for the experiments (17 inches from the floor of the box). The animals were exposed to an aerosol with 1.5 x 109 i 102 organisms during a time interval of one hour, (.25 x 109 i 102 per minute) using a culture of M. phlgl. After sufficient decay of the aerosol cloud (approx- imately one-half hour) the animals were removed from the aerosol chamber and immediately euthanatized. The lung was aseptically removed from the animals and divided into two parts, the anterior part consisted of the anterior half of the left lobe and the right superior and middle lobes, the posterior portion consisted of the poster- ior half of the left lobe and the right inferior and infer- ior medial lobes. The tissue was ground in a mortar with pestle and appropriate samples streaked on Middlebrook agar 37 plates, actidione, chloromycetin and erythromycin in Middle- brook plates and Dubos agar plates both prior to and after NaOH treatment of the ground tissues. A control group of 5 guinea pigs was euthanatized and the lungs removed and cultured in a similar manner. Due to the size of the aerosol chamber no attempt was made to maintain constant humidity. Therefore, any varia- tion from day to day in the relative atmospheric humidity could cause a change in the percent viability of the organ- isms in the aerosol and also possibly the particle size. However, during the course of the inoculations, the relative humidity remained above 70% due to the moisture present in the expired air from the animal. 4. Inoculation of Animals The calves in each isolation room were inoculated with a different Group-III isolant of bovine origin. The con- centration of the organisms in the culture medium was 1 x 108 per ml. The total exposure for each animal was approx- imately 1.5 x 109 1'102 organisms per hour since approximate- ly 15 ml. of culture medium were used per hour. This amount was more or less arbitrarily selected based principally on previous reports and limited data collected from the pilot studies. Three calves from an isolation room were inoculated each day with one culture. This allowed the Operator time to 38 disinfect the aerosol chamber, and the contaminated working area, sterilize the atomizer-venturi apparatus and change the filter before inoculation of the next group of animals. Two persons composed the team necessary to inoculate the calves. The Operators wore white coveralls, disposable cap and mask, rubber apron, boots and surgical gloves while operating the aerosol chamber. After the completion of inoculating the three calves, each Operator removed his contaminated attire and showered thoroughly. Four corner tie rOpes were attached at the entrance of the box in order to fix the position of the external nares of the calf in proximity to the aerosol cloud. The aerosol chamber was placed outside the isolation room door. The three animals within the isolation room were haltered and each injected with 400 mg. promazine* intravenously approx- imately ten minutes before placing them in the aerosol cham- ber. Each calf was backed into the chamber and the four tie ropes attached to the halter. The secondary air flow was allowed to run constantly to maintain a negative pressure away from the aerosol chamber door, thus reducing the danger of aerosol contamination to the Operator. After the calf was secured in position and the door was closed and sealed, the primary air flow was turned on and the timer set for one hour. Following this interval, the primary air flow was stopped and the aerosol cloud was *Sparine, Wyeth Laboratories, Inc., Philadelphia, Pennsylvania. 39 allowed to decay for é hour before Opening the aerosol chamber. With minimum contact between the Operator and the aerosol chamber and calf, the tie ropes were unfastened and a lead rOpe tied to the halter. The calf was led from the aerosol chamber into the isolation room and tied to a wall ring. A second calf was backed into the aerosol chamber and the process repeated. The skin of the infected animal, with the exception of the head, was disinfected, using a high pressure (600 pounds per square inch) Sprayer* containing orthophenylphenol disinfectant-detergent (0.5%). The head of the animal was scrubbed with a brush and the same disin- fectant. After approximately 15 minutes the animal was flushed with warm running water. 5. Clinical Examinations The same observations as described under the intra- uterine experiment were followed, except that rectal exam— inations were not completed. In addition, lung auscultation was completed in order to detect and follow the clinical course of any pulmonary disturbance. 6. Tuberculin-Test Technique The tuberculin tests were completed using identical procedures as outlined under the intrauterine experiment. *Liquid Brush, Kleen King, Britt Teck Corporation, Britt, Iowa. 40 7. Necropsy Technique The technique described under the intrauterine experi- ment was followed. 8. Bacteriologic Technique The same bacteriologic techniques were followed for examination of the tissues as was described in the intra- uterine experiment. However, the following lymph nodes and organs were submitted for bacteriologic examination: A. Right and left submaxillary, right and left medial and lateral retropharyngeal and right and left parotid lymph nodes. B. Right and left bronchial (including the right apical bronchia1)and anterior and posterior mediastinal lymph nodes. C. Mesenteric, colic and forestomach lymph nodes. E. Lung. H. Intestine (ileum). I. Liver and spleen. 9. HistOpathologic Technique The techniques were similar to the methods described in the intrauterine experiment. In addition to the tissues mentioned above, the follow- ing were fixed in 10% buffered formalin for histopathologic examination: right and left prescapular, right and left axillary, hepatic, right and left internal iliac, right and left popliteal, right and left superficial inguinal, right 41 and left deep inguinal, right and left ischiatic, and right and left prefemoral lymph nodes; heart; kidneys and brain. TABLE 1. PAGINATION OF RESULTS ACCORDING TO ANIMALS AND CULTURES USED Intrauterine Experiment Animal Culture Page No. No. NO. 74 51C-0 3 75 510-0 46 77 510-0 49 73 680-0 54 7O 68C-O 58 71 680-0 65 78 SOB-O 73 79 BOB-O 75 80 SOB-O 76 Aerosol Experiment Animal Culture Page NO. NO. NO. 91 510-0 79 84 510-0 84 90 510-0 87 82 68C-O 90 86 68C-O 94 83 680-0 101 89 SOB-O 112 87 SOB-0 113 88 SOB-O 114 42 RESULTS A. Intrauterine Experiment 1. Heifers Inoculated with Culture BlC-O H81fer 74 '" To B. Lab. NO. 90 "' 63 Clinical History On the 21st day following inoculation a yellow purulent vaginal discharge was noted. The highest temperature after inoculation was 102.4 F. On the 42nd day after inoculation a rectal examination revealed the following: 1) no indica- tions of pregnancy, but both horns of the uterus were enlarged and the walls thickened, 2) the left ovary was larger than the right and also contained a developing fol- licle, 3) the cervix was slightly enlarged. The animal continued to lose condition from the time of inoculation until necropsy. Necropsy Findings (62 days after inoculation) Guernsey heifer, approximately 24 months old, fair condition, weight 450 lbs. There were several circumscribed, raised plaques of red- dish fibrinoid material scattered over the surface of the omentum. These lesions varied from .5 to 1.5 cm. in diameter. 43 44 Three of the mesenteric lymph nodes contained a few small caseous lesions (from 2 to 3 mm. in diameter) within the cortex. The left supramammary lymph node was markedly enlarged (10 x 7 x 4.5 cm.) and contained a large encapsulated cavity (4 x 3 x 3 cm.) filled with purulent material. The wall of the uterus was thickened and the mucosal surface was covered with numerous raised nodules. There was yellowish gritty material (from 1 to 3 mm) in a few of the lesions. The left ischiatic lymph node was markedly enlarged (3 x 3 x 3 cm. in diameter) and contained a caseous focus approximately 1 cm. in diameter. Histopathologic Findings The lesions of the left ischiatic lymph node consisted of several irregularly shaped to circular foci of macrophages and/or epithelioid cells (hereafter often referred to as a granulomatous reaction) with giant cell formation. In many of the inflammatory areas, central caseation necrosis was present and there was a mild polymorphonuclear infiltration. The lesion of the left supramammary lymph node consisted of a large area of caseation necrosis surrounded by a granu- lomatous reaction with giant cell formation and early calci— fication. Peripherally, there was some fibroblastic proliferation. 45 The colic lymph node had an area of diffuse leukocytic infiltration predominantly in the cortex. The lumbar lymph node had several microscopic foci of granulomatous reaction some of which had early central necro- sis. The submucosa of the intestine had several foci of necrosis surrounded by a mixed granulomatous reaction. The lamina pr0pria Of the uterus had several develOping tuberculous lesions characterized by granulomatous reaction with giant cells. The mucosal epithelium over some of these lesions had undergone necrosis. Acid-fast bacilli were demonstrated in the lesions of all the lymph nodes and organs studied except the colic and lumbar lymph nodes and the intestine. Bacteriologic Findings Acid-fast bacteria were recovered from (A) the pool of the right and left submaxillary, right and left parotid, right and left medial and lateral retropharyngeal lymph nodes; (B) the pool of the right and left bronchial and the anterior and posterior mediastinal lymph nodes; (C) the pool of the mesenteric, colic and hepatic lymph nodes; (J) the pool of the right and left internal iliac, right and left deep inguinal and lumbar lymph nodes; (G) the right and left supramammary lymph nodes; (I) the pool of the liver and spleen and (K) the uterus. 46 Group-III mycobacteria were isolated from 4 out of 10 (4/10) tubes from (A), from 2/10 tubes from (B), 2/10 tubes from (C), 6/10 tubes from (J), 3/10 tubes from (G), 10/10 tubes from (K) and 2/10 tubes from (I). Numerous colony forms were isolated similar to the original culture. One appeared to be a Group II which may be an orange-smooth variant of the original culture. Heifer 75 - T. B. Lab. No. 116 - 63 Clinical History A purulent vulvar discharge was first Observed approx- imately 2 weeks after inoculation. On rectal examination 43 days after inoculation, there was no evidence of preg- nancy and the cervix, uterus and ovaries were apparently nor- mal. The rectal temperature remained normal. There was a progressive loss of weight starting approximately 2 months after inoculation. NecrOpsy Findings (124 days after inoculation) Guernsey heifer, approximately 24 months of age, fair condition, body weight approximately 800 lbs. The posterior mediastinal lymph nodes had four circum- scribed foci Of yellowish caseous material 2 mm. in diameter. The anterior mediastinal lymph node contained three circumscribed foci of yellowish caseous material from 1 to 3 mm. in diameter. The right and left bronchial lymph nodes had several Small caseous foci (1 to 3 mm. in diameter). 47 There were several reddish-colored plaques of solid tissue from 5 to 10 mm. in diameter on the serosal surface of the spleen. The wall of the uterus was slightly thicker than nor- mal and the mucosal surface had several yellowish-gray nodules (varying in diameter from 2 to 6 mm.) scattered throughout the mucosa. Many of the lesions had a central core of caseous material. The left and right supramammary lymph nodes were marked- ly enlarged and had four circumscribed foci of caseous mater- ial from 2 to 8 mm. in diameter. One of the colic lymph nodes had a circumscribed area of yellowish discoloration revealed on incision. There were several raised, reddish plaques surrounding the ovaries and fimbriae. The surface of the omentum was extensively covered with reddish-gray plaques from 2 to 15 mm. in diameter. Histopathologic Findings The cortical regions of the right and left bronchial lymph nodes had large areas of caseation necrosis and early calcification surrounded by granulomatous reactions with giant cells. There was an incomplete fibrous connective tissue encapsulation of the lesions. The lesions of the anterior mediastinal lymph node consisted of several circumscribed foci of granulomatous reaction with giant cells, central caseation and early 48 calcification. Most of the advanced lesions were partially encapsulated. Confluent with the more advanced lesions were areas of non-caseating granulomas. The lesions of the posterior mediastinal lymph node consisted of two circumscribed foci of caseation necrosis surrounded by granulomatous reaction. Also in the inflamma- tory area were a moderate neutrOphilic infiltration and some giant cell formation. A microscopic focus of macrophages was noted in the vicinity of a portal triad of the liver. The lamina propria of the uterus had extensive areas of lymphocytic and monocytic infiltration. In some areas there was necrosis involving the surface epithelium and some of the adjacent glands. There were a few circumscribed foci of granulomatous reaction with central caseation necrosis be- neath the epithelial surface. Occasionally, a similar reac- tion involved uterine mucosal glands accompanied by marked neutrOphilic infiltration and loss of glandular epithelium. There was some increase in fibrous tissue in the lamina propria. The lesions of the fimbriae consisted of extensive areas of non-caseating granulomas with giant cell formation. The cortical regions of the right and left supramam- mary lymph nodes had few circumscribed foci of caseation necrosis, each surrounded by a zone of granulomatous reac- tion with giant cells. There was some fibroblastic prolif- eration around the lesions. 49 The right ischiatic lymph node had a circumscribed focus of granulomatous reaction with early central casea- tion necrosis. There was also some leukocytic infiltration, giant cell formation and some peripheral fibroblastic prolif- eration. Acid-fast bacilli were demonstrated in all the lesions of the lymph nodes and organs studied except the liver. Bacteriologic Findings Acid-fast bacteria were recovered from (B) the pool of the right and left bronchial and the anterior and posterior mediastinal lymph nodes; (C) the pool of the mesenteric, colic and hepatic lymph nodes; (E) the lung; (G) the pool of the right and left supramammary lymph nodes; (J) the pool of the right and left internal iliac, right and left deep inguinal and lumbar lymph nodes; (I) the pool of the liver and Spleen and (K) the uterus. Group-III mycobacteria were isolated from 10 out of 10 (10/10) tubes from (B), 2/10 tubes from (C), 2/10 tubes from (B), 9/10 tubes from (G), l/lO tubes from (J), l/lO tubes from (I) and 5/10 tubes from (K). Heifer 77 - T. B. Lab. No. 151 - 63 Clinical History Forty-three days after inoculation, a rectal examina- tion revealed the following: 1) adhesions of the left ovary, fimbria and Fallopian tube, 2) an apparently normal uterus, 50 and 3) no signs of pregnancy. There was some loss of body condition starting approximately 59 days after inoculation. A purulent vulvar discharge was first noted 146 days after inoculation. Rectal temperature was normal throughout the experiment. A second rectal examination was completed 167 days after inoculation and the following were noted: 1) ad- hesions between the right fimbria and ovary, 2) a follicu- lar cyst on the left ovary and adhesions between the latter and fimbria, 3) some fluid in both horns of the uterus. NecrOpsy Findings (176 days after inoculation) Guernsey heifer, approximately 29 months old, fair condition, body weight 700 lbs. The cortical region of the right ischiatic lymph node contained a few circumscribed foci of caseous material from .5 to 2 mm. in diameter. The left ischiatic lymph node had a few circumscribed caseous lesions in the cortical region from 1 to 5 mm. in diameter. The right and left medial retrOpharyngeal lymph nodes were enlarged, one was 7 x 4 x 4.5 cm. and the other 5 x 4 x 4.5 cm. The lymph node contained multiple circumscribed foci of caseous material from 2 to 5 mm. in diameter. The posterior mediastinal lymph node had several cir- cumscribed caseous foci from 2 to 4 mm. in diameter. 51 The cortical region of two lymph nodes of the lumbar chain had several circumscribed foci of caseous material from 2 to 4 mm. in diameter. The internal iliac lymph nodes were markedly enlarged and contained multiple circumscribed foci of caseous materu ial from 1 to 4 mm. in diameter. The serosal surface of the spleen and particularly the omentum had several raised, reddish circumscribed plaques. When incised, the lesions were solid throughout. Multiple fibrous adhesions were noted between the serosal surface of the intestine and body wall, and between the liver and diaphragm. One of the supramammary lymph nodes was markedly en- larged (6 x 2 x 3 cm.). Incision revealed several soft, yellow, caseous foci from 4 to 15 mm. in diameter. One of the mesenteric lymph nodes had a discrete non- caseous focus of yellowish discoloration (3 mm. in diameter) in the cortex. The mammary gland was enlarged, firm and had multiple circumscribed foci of caseous material throughout from 2 to 7 mm. in diameter. The mucosal surface of the intestine had a few raised circumscribed nodules which contained greenish caseous mater» ial. The right and left horns of the uterus were slightly enlarged and the mucosal surface was reddened and contained 52 numerous yellowish, circumscribed foci of caseous material approximately 2 mm. in diameter. Histopathologic Findings The lesions Of the uterus were characterized by circum- scribed foci of epithelioid cells, with an occasional giant cell, within the lamina pr0pria. In some cases, the lesions were surrounded by lymphocytic infiltration. The lesions of the supramammary lymph node consisted of several circumscribed and confluent foci of caseation necro— sis surrounded by a narrow zone of granulomatous reaction with giant cells. The more advanced lesions were partially encapsulated. In some of the foci of inflammation the cas- eous material was moderately infiltrated with neutrOphils and calcium. The right and left internal iliac lymph nodes had several circumscribed foci of granulomatous reaction, some of which had early central caseation necrosis. The inflam- matory process contained numerous giant cells and early calcification. The more advanced lesions were partially encapsulated. The right ischiatic lymph node had a few small circum- scribed foci of caseation necrosis with early calcification. These were surrounded by narrow zones of granulomatous reac- tion with giant cell formation. The granulomas were encap- sulated. 53 The left ischiatic lymph node had a few circumscribed foci of granulomatous reaction with giant cells and central caseation necrosis. Some of the lesions were encapsulated. The right and left medial retropharyngeal lymph nodes had several large circumscribed foci of caseation necrosis surrounded by narrow zones of epithelioid and giant cells. Around the periphery of the lesions were incomplete fibrous connective tissue capsules. The cortical region of the mediastinal lymph node had a few small circumscribed foci of granulomatous reaction with some giant cells, central caseation necrosis and early calcification. The lumbar lymph nodes had a few small circumscribed foci of granulomatous reaction with central caseation necro- sis and early calcification. The inflammatory reaction con— tained giant cells and was in the cortical region adjacent to the capsule. The cortical region of one colic lymph node had a small circumscribed focus of granulomatous reaction with giant cells, central caseation necrosis and calcification. Periph- erally, there was an incomplete fibrous connective tissue capsule. The left prescapular lymph node had a few circumscribed foci Of granulomatous reaction with central caseation necro~ sis and early calcification. Giant cell formation was present in the inflammatory reaction and there was some peripheral fibrous connective tissue proliferation. 54 The lamina pr0pria of the intestine had a marked diffuse granulomatous reaction and giant cell formation. There were also a marked neutrOphilic infiltration and necrosis of much of the intestinal epithelium. There was fibrous connective tissue proliferation in some parts of the lamina propria. Acid-fast bacilli were present in all lesions of the lymph nodes and organs examined except the uterus. Bacteriologic Findings Acid-fast bacteria were recovered from (A) the pool of the right and left submaxillary, right and left parotid, right and left medial and lateral retropharyngeal lymph nodes; (C) the pool of the mesenteric, colic, and hepatic lymph nodes; (E) the lung; (G) the pool of the right and left supramammary lymph nodes; (J) the pool of the internal iliac, the right and left deep inguinal and lumbar lymph nodes; (1) the pool Of the liver and spleen and (K) the uterus. Group-III mycobacteria were isolated from ten out of 10 (10/10) tubes from (A), 6/10 tubes from (0), 1/10 tubes from (E), 9/10 tubes from (G), 8/10 tubes from (J), 3/10 tubes from (I) and 5/10 tubes from (K). 2. Heifers Inoculated with Culture 68C-0 Heifer 73 - T. B. Lab. NO. 89 - 53 Clinical History On the 42nd day after inoculation, rectal examination revealed the following: 1) no indication of pregnancy; 55 2) follicular develOpment on the left ovary; 3) a thickened and enlarged uterus. The animal had progressively lost weight following inocu ulation and the rectal temperature had not been observed above 103 F. Approximately 10 days after inoculation, the animal had a purulent vulvar discharge which persisted until the time of necropsy. NecrOpsy Findings (62 days after inoculation) Holstein heifer, approximately 22 months Old, poor condition, body weight 400 lbs. Two of the mesenteric lymph nodes contained several circumscribed foci, from 1 to 4 mm. in diameter, which con- tained a greenish, caseous material. The cortical region Of one lymph node had a solid yellow focus 3 mm. in diameter and another an encapsulated abscess (8 mm. in diameter). The mucosal surface of the uterus had multiple, raised, grayish-white, nodular foci (Fig. 3) from 2 to 6 mm. in diameter which were solid throughout. A gritty material was demonstrated in some lesions. The mucosal lining was hypera emic and covered with purulent exudate. Scattered lesions were found over the serosal surface of the omentum and were characterized by multiple, circum- scribed, raised, reddish fibrinous plaques from .5 to 1.5 cm. in diameter. The right supramammary lymph node contained a circum— scribed focus of caseous material .5 x 1 cm. in diameter. u5?9.c « Fig. 3. Segment of uterus from heifer 73, 62 days after inoculation with culture 68Cm0. Note raised nodules on mucosal surface. 56 57 The left supramammary lymph node contained several small circumscribed foci of caseous material from 2 to 3 mm. in diameter. Histopathologic Findings The mesenteric lymph nodes had large, irregularwshaped and circular areas of caseation necrosis and early calcifica- tiOn surrounded by a granulomatous reaction with giant cell formation. There was a marked leukocytic infiltration around some reactive areas. Several noncaseating granulomas (daugh~ ter tubercles) extended from the more advanced lesions. The right medial retrOpharyngeal lymph node had several foci of granulomatous reaction with few giant cells and some early central caseation necrosis. The left medial retropharyngeal lymph node had several microscopic foci of granulomas, some of which had early cen- tral caseation necrosis. The right and left supramammary lymph nodes had several caseating and noncaseating granulomas. The larger lesions were encapsulated. The intestinal submucosa had several foci of a mixed granulomatous reaction. The typical lesion consisted of a central area of necrosis surrounded by a granulomatous reac- tion, infiltrated with numerous eosinOphils. Cross sections of nematodes were Observed in some of the lesions. The omental lesions consisted of granulation tissue within which were micrOSOOpic foci of tuberculous granulomas with leukocytic infiltration. 58 The uterine lesions consisted of foci of developing tuberculous inflammation in the lamina propria beneath the surface epithelium. There was a granulomatous reaction with some evidence of focal necrosis and leukocytic infiltration. Acid-fast bacilli were demonstrated only in the mesen- teric lymph nodes and the uterus. Bacteriologic Findings Acid-fast bacteria were recovered from (A) the pool of the right and left submaxillary, right and left parotid, »right and left medial and lateral retropharyngeal lymph nodes; (C) the pool Of the mesenteric, colic and hepatic lymph nodes, a lesion from (C); (E) the lung; (G) the pool of the right and left supramammary lymph nodes; (J) the pool of the internal iliac, right and left deep inguinal and lum- bar lymph nodes; (H) the intestine and (K) the uterus. Group-III mycobacteria were isolated from nine Out of 10 (9/10) tubes from (A), 4/10 tubes from (C), 8/10 tubes from a (C) lesion, 2/10 tubes from (E), 6/10 tubes frOm (G), 2/10 tubes from (J), 1/10 tubes from (H) and 7/10 tubes from (K). Heifer 7O - T. B. Lab. NO. 117 - 63 Clinical History A loss of body condition was apparent approximately 42 days after inoculation and persisted until necrOpsy. On the 43rd day after inoculation a rectal examination revealed the following: 1) no signs of pregnancy; 2) no develOping ovarian follicles or corpora lutea; 3) enlargement of both horns of the uterus, the right horn appearing larger than the left. A purulent vulvar discharge started approximately 83 days after inoculation and persisted until necropsy. The highest rectal temperature recorded after inoculation was 103 F. Necropsy Findings Guernsey heifer, approximately 24 months, fair condition, body weight 700 lbs. One of the enlarged medial retropharyngeal lymph nodes had several caseous foci (up to 1 mm. in diameter). The other lymph node had similar foci from 2 to 5 mm. in diamem ter. One of the lateral retrOpharyngeal lymph nodes was enlarged and, on incision, had a circumscribed focus of yellowish-white discoloration (approximately 5 mm. in diame~ ter) with streaks of caseous material. The liver had 2 circumscribed foci of caseous material which were 2 mm. in diameter. In another area of the liver, there was a focus of yellowish discoloration approximately 3 mm. in diameter. The spleen contained several raised, reddish plaques of solid-appearing tissue over the border and surface. In some instances, the lesions extended into the parenchyma of the organ. 60 Two lymph nodes of the mesenteric group contained les- ions. One had a focus of greenish-yellow material 5 mm.in diameter and the other contained a focus of yellowish cas- eous material 3 mm. in diameter. The right and left internal iliac lymph nodes were enlarged and contained several foci of caseocalcareous mater~ ial 2 to .5 mm. in diameter. One of the two supramammary lymph nodes was markedly enlarged and contained multiple circumscribed foci of yellow= ish caseocalcareous material from 2 to 15 mm. in diameter. The other was slightly enlarged and contained two foci of caseocalcareous material (2 and 8 mm. in diameter respective- 1y). The wall of the uterus appeared to be thickened. There were raised nodules on the mucosal surface some of which had caseous centers and others a yellowish discoloration. There was a purulent exudate over the surface of the mucosa. The cervix was thickened and contained a yellowish circumscribed nodule on the mucosal surface. The omentum was markedly thickened (up to 2 cm. in some areas) and was extensively covered with raised, reddish nodules (Fig. 4) of tissue which were solid throughout. Sims ilar lesions occurred on the serosal surface of the pelvic cavity. Histopathologic Findings The lesions of the submaxillary lymph nodes consisted of circumscribed foci and confluent areas of granulomatous 61 _ IlIIII IIII,.U_I,HIIIII IIIIIIIII IIIIIIIII IIIIIIIII IIIIIIIII IIIIIIIII IIIII Fig. 4. Omentum from heifer 70,124 days after inoculation with culture 680- 0. Note the multiple, raised, irregularly shaped nodules in the serosal surface (arrows). c . “J, , . J. '1 ‘ ‘. é , ‘ - q o. 3328‘s.. (35...”1i": ‘ 3134* Fig. 5. Omentum from heifer 70,124 days after inoculation with culture 680- 0. Note the area of necrosis, giant cells, surrounded by lymphoid and epithelioid cells. New Fuchsin--H & E. x187. 62 reaction with giant cells and some central caseation necroe sis. Extending from many of the larger lesions were solid areas of granulomatous reaction typical of "daughter tubers cle" formation. In some areas, the advanced lesions were partially encapsulated. The medial retrOpharyngeal lymph node had irregular— shaped and circular foci of granulomatous reaction with giant cells scattered throughout the parenchyma. Many of the les- ions were coalescing and areas of central caseation necrosis were evident. The posterior mediastinal lymph node contained extensive areas of coalescing granulomas with giant cell formation. In some areas there was evidence of early caseation necrosis. The cortical region of the lateral retropharyngeal lymph node had circumscribed foci of granulomatous reaction. There were numerous giant cells and early central caseation was evident. The mesenteric lymph nodes had several partially encap- sulated foci of caseation necrosis and early calcification surrounded by a narrow zone of epithelioid cells and a few giant cells. In another part of the node there was a leuko- cytic infiltration (predominantly eosinophils). The omental lesions consisted of solid coalescing areas of granulomatous reaction with giant cells and some areas of early central caseation necrosis. There was a narrow incom- plete fibrous connective tissue capsule partially surround- ing the lesion. 63 The internal iliac lymph nodes had extensive areas of granulomatous reaction with giant cells throughout most of the parenchyma. In some areas there was confluence of the inflammatory reaction with caseation necrosis and calcifica- tion. The right deep inguinal lymph node had a few circum- scribed microscopic foci of granulomatous reaction and neutro- philic infiltration. The left deep inguinal lymph node contained a small circumscribed focus Of granulomatous reaction in the cortex and adjacent to the capsule. There was central caseation necrosis and giant cell formation. The lumbar chain of lymph nodes had numerous circum- scribed foci of non-caseating granulomas with giant cells. In some of the lesions early caseation necrosis and calcifi- cation were evident. The submucosa of the intestine had an area of caseation necrosis infiltrated with neutrOphils and surrounded by a mixed granulomatous reaction (macrOphages and neutrOphils). The uterus had a marked granulomatous reaction with some giant cells and central caseation extending from the epithe- lial surface into the lamina propria. Much of the columnar epithelium over these lesions had undergone necrosis and was absent in some areas. There was considerable lymphocytic and neutrOphilic infiltration in the lamina propria particu- larly in the immediate vicinity of some of the uterine muco- sal glands. 64 The left and right supramammary lymph nodes had circum» scribed foci and confluent areas of extensive granulomatous reaction and central caseation necrosis. There were numer- ous giant cells and early calcification throughout the reac- tion. Some of the lesions were partially encapsulated. Acid-fast bacilli were demonstrated in the lesions of all the lymph nodes and organs examined except the mesenter- ic and left deep inguinal lymph nodes and the intestine. Bacteriologic Findings Acid-fast bacteria were recovered from (A) the pool of the right and left submaxillary, right and left parotid, right and left medial and lateral retropharyngeal lymph nodes; (B) the pool of the right and left bronchial and the anterm ior and posterior mediastinal lymph nodes; (0) the pool of the mesenteric, colic and hepatic lymph nodes; (E) the lung; (G) the pool of the right and left supramammary lymph nodes; (H) the intestine; (J) the pool of the internal iliac, the right and left internal deep inguinal and lumbar lymph nodes; and (K) the uterus. Group-III mycobacteria were isolated from seven out of 10 (7/10) tubes from (A), 7/10 tubes from (B), 3/10 tubes from (C), 6/10 tubes from (E), 5/10 tubes from (G), 2/10 tubes from (H), 7/10 tubes from (J) and 3/10 tubes from (K). 65 Heifer 71 - T. B. Lab. NO. 150 - 63 Clinical History The animal had some loss of condition and a purulent vulvar discharge starting approximately 30 days after inocu- lation. The rectal temperature remained normal during the experiment. Approximately 43 days after inoculation a rectal examination revealed the following: 1) no detectable signs of pregnancy, 2) no developing ovarian follicles or corpus luteum, and 3) enlargement of the uterine horns with fluid in the left horn. At 72 and 146 days after inoculation there was a notice» able swelling of the vulva and the left anterior and poster- ior quarter of the mammary gland reSpectively. The animal continued to lose weight and condition until necrOpsy. A second rectal examination was completed 169 days after inoculation and the following abnormalities were noted: 1) enlargement Of both uterine horns with palpable nodules, and 2) the left ovary was enlarged and firm. Necropsy Findings (176 days after inoculation) Holstein heifer, approximately 30 months of age, extremely poor condition, body weight 800 lbs. The left ischiatic lymph node was 4 x 3 x 4 cm. and contained multiple confluent foci of caseous material. The cortical region of the right ischiatic lymph node contained a circumscribed focus of caseous material 3 to 4 mm. in diameter. There were several noncaseous, raised, reddish fibrin- ous plaques on the surface Of the omentum from 5 to 15 mm. in diameter. The right anterior and posterior quarters of the mammary gland was markedly enlarged, cut with difficulty and had ex“ tensive circumscribed foci of caseous and purulent material throughout the parenchyma. The lesions were from 3 to 20 mm. in diameter. The supramammary lymph nodes were markedly enlarged, one was 8.5 x 7 x 5 cm. and the other 6 x 2.5 x 5 cm. There were circumscribed foci and confluent areas Of caseous mater- ial from 2 mm. in diameter to 2 x 4 x 4 cm. The uterus was enlarged to approximately 7 cm. in diame- ter in some areas. The mucosa was congested and the uterine walls were markedly thickened. There were extensive areas of irregularly-shaped nodules some of which had caseous cen- ters (Fig. 6). There were similar lesions on the serosal surface of the uterus. The lumen of the uterus contained approximately 2 L. of purulent material. The vulva was markedly swollen, firm and had several focal areas of yellow caseous material and purulent exudate from 5 to 15 mm. in diameter. The ovaries were normal except for a few fibrinous adhesions on the surface. The posterior mediastinal lymph nodes had several cir~ cumscribed foci of caseous material from 2 to 10 mm. in diam- eter. 67 Fig. 6. Uterus from heifer 71, 176 3;}; after inocula- tion with culture 680-0. Note the extensive tuberculous process with caseation (arrows) in the thickened walls. .,I *3 5 s69 M11. 01334 Q '4‘) .47"-*3’. at 3 4p:% *3 . f! 6 ya .:§1¥ o ox, . ’1 V .w 0 b. ‘1‘}. . .,.e-.~.:,,.;t.e:w ' * 35:33 'fi‘“:~I=vC-'w .fié. ' - ‘ :0 401"} l” 19g‘o.fi,.v g}' j ~ - “"1. Kg.“ 0? .731“;- b}? ‘( ' ~ . I 3:0 ?;"'u,:':i§ '. a. '«6 "or a -' N. r‘ A' ‘A ‘ ;,.‘.; I. 3.31 ' .. :g, fitfiifi: 3.5.0‘ _-‘ 1",'.‘ - 34-3 whim...» , . «‘11.»..3: .33“. Fig. 7. Uterus from heifer 71, 176 days after inocula- tion with culture 680-0. Note the mucosal glands (arrow) filled with neutrOphils, epithelioid cells in the lamina propria, and suppuration (l) in the area of the destroyed epithelium of the endometrium. New Fuchsin--H & E. x187. 68 The right deep inguinal lymph node was slightly enlarged and, in the cortical region, there were several irregularly shaped to circular foci of caseous material from 2 to 4 mm. in diameter. The left deep inguinal lymph node was markedly enlarged (8 x 4.5 x 5 cm.) and had several circumscribed caseous foci from 3 to 35 mm. in diameter. Two lumbar lymph nodes were enlarged (4.5 x 3.5 x 3.5 cm.) The lesions consisted of circumscribed foci of caseous material which extended from the cortex into the medulla by linear streaks. The right and left internal iliac lymph nodes were en- larged to 6.5 x 4 x 6 cm., 3 x 5.5 x 3.5 and 3.5 x 1.75 x 1.5 cm. in diameter. There were multiple irregularly-shaped and circular foci of caseous material in the cortex from 2 to 15 mm. in diameter. The left and right parotid lymph nodes were slightly enlarged. Circumscribed foci of caseous material were present. The medial retrOpharyngeal lymph nodes were markedly enlarged; one was 10 x 7 x 6 cm. and the other 6 x 9 x 4 cm. Multiple irregularly-shaped to circular foci of caseous material were found throughout the lymph nodes. One of the anterior mediastinal lymph nodes had a circumscribed focus of caseous material which was 6 mm. in diameter. 69 The left bronchial lymph node had a few circumscribed foci (2 to 4 mm. in diameter) of caseous material. The lung parenchyma had a few scattered circumscribed foci of caseous material from 3 to 5 mm. in diameter. Histopathologic Findings The lesions of the left bronchial lymph node consisted of extensive areas of caseation necrosis surrounded by a nar- row zone of granulomatous reaction with some giant cells. The caseous centers had undergone early calcification and there was some neutrOphilic infiltration. The lumbar lymph nodes had circumscribed and confluent areas of caseation necrosis surrounded by a granulomatous reaction with pronounced giant cell formation. The caseous centers were undergoing early calcification. The lesion of the left ischiatic lymph node consisted of large circumscribed foci and confluent areas of caseation necrosis surrounded by a granulomatous reaction with giant cells. The right ischiatic lymph node had a circumscribed focus of caseation necrosis with early calcification. The lesion was surrounded by a zone of epithelioid cells with a few giant cells and was partially encapsulated. Most of the posterior mediastinal lymph nodes had ex- tensive confluent areas of caseation necrosis surrounded by a granulomatous reaction with abundant giant cells and periphe eral connective tissue proliferation. The inflammatory reac- tion also contained some neutrophils. 70 The lesions in the mammary gland consisted of multiple circumscribed foci and confluent areas of granulomatous reac- tion. Some of the lesions had central caseation necrosis and neutrOphilic infiltration. The uterine lesions were characterized by a marked in- filtration of the lamina pr0pria with epithelioid cells and neutrophils (Fig. 7) and formation of giant cells (Fig. 8). In some areas there was early caseation necrosis and early calcification. The surface epithelium and epithelium of the mucosal glands were absent in many areas. The pulmonary lesions were partially encapsulated and characterized by coalescing circumscribed foci of caseation necrosis surrounded by a granulomatous reaction with giant cells. In many areas there was extension of the lesions by noncaseating granulomas. The left deep inguinal lymph node contained multiple circular and irregularly-shaped foci of granulomatous reac- tion with giant cells, central caseation necrosis and early calcification. The parotid lymph nodes contained multiple circumscribed foci of granulomatous reaction and neutrOphilic infiltration. The larger foci had central caseation necrosis and calcifica- tion. The lesions were partially encapsulated and primarily located in the cortical region adjacent to the capsule. The internal iliac lymph node had circumscribed areas of caseation necrosis surrounded by a granulomatous reaction Fig. 8. Another area of the uterus shown in Fig. 7. Note the epithelioid cells, several giant cells (arrows) mixed with leukocytes. New Fuchsin--H & E. x187. 71 72 with some giant cells and neutrophils. In some parts of the caseous centers early calcification was noted. The lesions in the right deep inguinal lymph node con— sisted of circumscribed foci of caseation necrosis surrounded by epithelioid cells with giant cell formation. In some parts of the caseous centers early calcification was present. The posterior mediastinal lymph node had circumscribed and coalescing foci of caseation necrosis and early calcifica- tion surrounded by granulomatous reactions with giant cells. Extending from these lesions were solid nests of epithelioid cells typical of daughter tubercle formation. The medial retrOpharyngeal lymph nodes had circumscribed and coalescing foci Of caseation necrosis surrounded by a granulomatous reaction with giant cell formation. The lesions in the left and right supramammary lymph nodes consisted of several circumscribed foci of granulomas with some giant cells and central caseation necrosis. The lesions were primarily located in the cortical region adja- cent to the capsule. Acid-fast bacilli were demonstrated in lesions in all the lymph nodes and organs examined. Bacteriologic Findings Acid-fast bacteria were recovered from (A) the pool of the right and left medial and lateral retropharyngeal lymph nodes; (B) the pool of the right and left bronchial and the anterior and posterior mediastinal lymph nodes; (C) the pool 73 of the mesenteric, colic and hepatic lymph nodes; (E) the lung; (G) the pool of the right and left supramammary lymph nodes; (J) the pool of the right and left internal iliac, right and left deep inguinal and lumbar lymph nodes; (1) the pool of the liver and spleen and (K) the uterus. Group-III mycobacteria were isolated from nine out of ten (9/10) tubes from (A), 9/10 tubes from (B), 1/10 tubes from (c), 2/1o tubes from (E), 10/10 tubes from (c), 10/10 tubes from (J), 1/10 tubes from (I) and 9/10 tubes from (K). 3. Heifers Inoculated with Culture 50B-0 Clinical History The rectal temperature remained normal during the exper_ iment. Some purulent discharge was observed once around the tailhead. Rectal examination was completed 42 days after inoculation and the following were noted: 1) enlargement of the posterior portion of the cervix; 2) enlargement of the left ovary with apparent adhesions between it and the fimbria; 3) enlargement of the left horn of the uterus; 4) no sign of pregnancy. NecrOpsy Findings (64 days after inoculation) Guernsey heifer, approximately 22 months old, good condition, weight 550 lbs. There were several raised, reddish, fibrinous plaques scattered over the surface of the omentum. The lesions were 74 solid and had a uniform color throughout. No other gross lesions were detected in this animal. HistOpathologic Findings The intestine had a focus of mixed granulomatous reac- tion in the submucosa. The center of this lesion had under- gone necrosis and there was moderate leukocytic infiltration around and within the lesion (many eosinOphils). A mesenteric lymph node had a focus of mixed granulo- matous reaction (some neutrophils). In another lymph node there was a diffuse neutrOphilic infiltration. The cortical region of the left deep inguinal lymph node had several foci of macrophages and an occasional giant cell. Acid-fast bacilli were not demonstrated in lesions of any of the lymph nodes and organs examined. Bacteriologic Findings Acid-fast bacteria were recovered from (A) the pool of the right and left submaxillary, the right and left parotid, the right and left medial and lateral retrOpharyngeal lymph nodes; (G) the pool of the right and left supramammary lymph nodes; (J) the internal iliac, the right and left deep in- guinal and lumbar lymph nodes; and (I) the pool of the liver and spleen. Group-III mycobacteria were isolated from seven out of 10 (7/10) tubes from (A), 6/10 tubes from (G), 9/10 tubes from (J) and 1/10 tubes from (I). 75 Clinical History The animal's temperature remained normal and no vulvar discharge was noted throughout the experiment. 0n the 42nd day after inoculation the following were noted on rectal examination: 1) no evidence of pregnancy; 2) both ovaries appeared smaller than normal; 3) the cervix was enlarged, particularly the posterior half; 4) the uterus appeared to be normal. The heifer continued to gain weight and was in excellent physical condition at the time of necropsy. Necropsy Findings (125 days after inoculation) Holstein heifer, approximately 26 months old, excellent condition, body weight 800 lbs. The uterine mucosa was congested and there was con- siderable mucus in the cervical area. There were small raised yellowish nodules (2 to 3 mm. in diameter) which were uniformly scattered on the surface of the uterine mucosa. NO caseous centers were noted in these areas. These findings probably represented normal caruncles and signs of impending GSLI‘LIS o Histopathologic Findings The tissue sections of the mesenteric lymph node had one microscopic focus of macrOphages and one giant cell. Acid-fast bacilli were not demonstrated. 76 No microscopic lesions were found in the uterus. Bacteriologic Findings Although attempted, no isolations of mycobacteria were made. Heifer 80 - T. B. Lab. NO. 155 - 63 Clinical History The heifer was in excellent condition and the rectal temperature remained normal throughout the experiment. A rectal examination was completed 43 days after inoculation and the following were noted: 1) the right uterine horn was enlarged; 2) no evidence of pregnancy; 3) the ovaries were normal. A second rectal examination was completed 169 days after inoculation and the following were noted: 1) the uterus was normal; 2) develOping follicle and corpus luteum on the left ovary; 3) the right ovary had questionable ad- hesions. NecrOpsy Findings (181 days after inoculation) Ayrshire heifer, approximately 30 months of age, excellent condition, body weight approximately 900 lbs. The uterus was of normal size for a nonpregnant heifer. The mucosal surface appeared to be slightly congested and contained numerous slightly elevated yellowish nodules (2 to 3 mm. in diameter) within the mucosa. These areas re- vealed no caseous centers. These may have represented uter- ine caruncles. 77 NO detectable gross lesions were found in the remaining lymph nodes or organs examined. HistOpathologic Findings No microscopic lesions were detected in any of the organs or lymph nodes, including the uterus. Acid-fast bacilli were not demonstrated. Bacteriologic Findings Acid-fast bacteria were recovered from (A) the pool of the right and left submaxillary, the right and left parotid, the right and left medial and lateral retropharyngeal lymph nodes; (B) the pool of the right and left bronchial and the anterior and posterior mediastinal lymph nodes; (C) the pool of the mesenteric, colic and hepatic lymph nodes; (E) the lung; and (K) the uterus. Group-III mycobacteria were isolated from 1 out of 10 (1/10) tubes from (A), 1/10 tubes from (B), 1/10 tubes from (C), 2/10 tubes from (E), and 1/10 tubes from (K). One isolant when injected intradermally into a guinea pig produced a 2 x 5 mm. ulcer in 3 weeks. 78 B. Aerosol Experiment 1. Potential Infectivity of the Aerosol Table 2 summarizes the results of the guinea pigs ex- posed to aerosol using a culture of M. legi. Acid-fast isolations indistinguishable from the inoculum were recovered before sodium hydroxide treatment. .No acid-fast isolations were made following sodium hydroxide treatment. Trials I and II were conducted with essentially identical techniques. TABLE 2. SUMMARY OF RESULTS OF BACTERIOLOGICAL EXAMINATION (ACID-FAST ISOLATIONS) OF LUNG TISSUE FROM GUINEA PIGS EXPOSED TO AEROSOL WITH A CULTURE OF M. PHLEI Lobes of Lung Infected Animals Controls Trial I Trial 11 l 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Ante- rior - — - - - + + + + + - - - - _ Poste- rior - - + - + + + + + + - - - - - -No acid-fast isolations. +Acid-fast recovered on one or more culture media. 79 2. Calves Exposed to Aerosol with Culture 5lC-0 Calf 91 - T. B. Lab. No. 131 - 63 Clinical History Approximately 21 days after inoculation the animal had a persistent soft cough, serOpurulent nasal discharge, a temperature of 103 F, and inappetance (feed consumption was approximately a third of normal). There was also some de- pression and loss of body weight. A diarrhea first occurred 34 days after inoculation and continued until the time of necrOpsy. The highest temperature after inoculation was 104 F. The calf continued to lose condition and develOped a marked depression, purulent nasal discharge, complete anorexia, persistent soft cough, moist rales and some ab- dominal breathing. In moribund condition, the animal was euthanatized and necropsy was performed. Necropsy Findings (44 days after inoculation) Jersey steer, 7 1/2 months Old, poor condition, weight 250 lbs. The posterior mediastinal lymph node was markedly enlarged (17 x 7 x 4 cm.) and was cut with some difficulty. It had extensive confluent yellowish-gray areas with streaks and circular foci of caseous material. The capsule was thickened. The left and right bronchial lymph nodes were reSpective- ly 9 x 4 x 7 and 8 x 6 x 5 cm. in diameter. The gross lesions were similar to the above. 80 The anterior mediastinal lymph nodes were markedly enlarged, one to 6 x 3 x 4 cm. and another to 9 x 4 x 4 cm. The gross lesions in the lymph nodes were similar to the above. Approximately 10% of the liver tissue had a yellowish mottled appearance and the lobular markings were very prominent. The intestine was markedly congested and there was excessive mucus on the mucosal surface. The lungs had solid areas of red hepatization in the posterior portions of the apical and cardiac lobes. There were patchy areas of whitish-gray and red hepatization scat- tered over the surface of the remaining lung tissue. There were numerous circumscribed foci of solid grayish discolora— tion and caseous material uniformly scattered throughout all lobes of the lungs (Fig. 9). They measured from 1 to 6 mm. in diameter. There was marked interstitial edema Of the lungs and mediastinum. The heart was slightly enlarged and rounded at the apex. There was considerable loss of adipose tissue with serous atrOphy, particularly around the base of the heart. Histopathologic Findings The cortical regions of the right and left submaxillary lymph nodes had several irregularly shaped to circular foci of caseation necrosis surrounded by granulomatous reaction with giant cells. Extending from these lesions into the 81 Fig. 9. Lung from calf 91, 44 days after aerosol exposure with culture 51C-O. There are several subpleural tuberculous lesions (arrows). - ' 'x- I .‘.; .. j '- ’ I , '41. edéaitrw ”Ia.,fl;_g ti 'u3h-a§8§§ Fig. 10. Lung from calf 91, 44 days after aerosol exposure with culture 510-0. Note bronchiole (arrows) filled with inflammatory exudate and surrounded by granulomatous process. New Fuchsin--H & E. x187. 82 deeper cortical and medullary areas were solid foci of granuw lomatous reaction (early tubercle formation). The right and left medial retropharyngeal lymph nodes had a few circumscribed foci of granulomatous reaction with giant cells and caseation necrosis in the cortical region adjacent to the capsule. The left ischiatic lymph node had areas of leukocytic infiltration and numerous multinucleated giant cells. Some of the blood and lymph vessels in the capsule and the subcap~ sular sinus were filled with inflammatory cells characterized by macrophages and leukocytic cells (predominantly neutron phils). The anterior and posterior mediastinal and right and left bronchial lymph nodes were almost obliterated by ex- tensive areas of necrosis surrounded by areas Of granuloma- tous reSponse with giant cell formation. The capsules of the lymph nodes were thickened by proliferation of fibrous connective tissue. The pulmonary lesions consisted of numerous circum- scribed foci of granulomatous reaction with central casea- tion necrosis. The lesions involved both alveoli and bronchioles (Figs. 10 and 11) of the lungs. The alveoli surrounding the lesions were filled with serous exudate and a few alveolar macrOphages. In some of the caseous centers hemorrhage was also noted. Fig. 11. Higher magnification of the respiratory bronchiole shown in Fig. 10. Note the epithelioid cells, lymphocytes and a few neutrophils. New Fuchsin--H & E. x400. 83 84 Acid-fast bacilli were demonstrated in the lesions of all the lymph nodes and organs examined except the left ischiatic lymph node. Bacteriologic Findings Acid-fast bacteria were isolated from (A) the pool of the right and left submaxillary, right and left parotid, right and left medial and lateral retropharyngeal lymph nodes; (B) the pool Of the right and left bronchial, anterior and posterior mediastinal lymph nodes and (E) the lung. Group-III mycobacteria were isolated from nine out of ten tubes (9/10) inoculated from (A), 9/10 tubes from (B) and 8/10 tubes from (E). Calf 84 - T. B. Lab. NO. 132 - 63 Clinical History Approximately 12 days after inoculation the animal had signs of some loss of condition, a soft moist cough and slight serOpurulent nasal discharge. The animal had an intermittent fever throughout the course of the experiment which reached a high of 104.6 F. The feed consumption had decreased approximately one-third due to partial anorexia and the animal was depressed. These signs persisted until necrOpsy. Approximately 40 days after inoculation the animal had a watery, fetid diarrhea and marked depression. Since death was anticipated the calf was euthanatized and necropsy performed. 85 Necropsy Findings (45 days after inoculation) Jersey steer, 6 1/2 months old, poor condition, weight about 250 lbs. The lungs were enlarged and had patchy areas of gray and red hepatization visible over the surface and throughout the incised parenchyma. The pulmonary parenchyma also had extensive irregularly shaped to circular solid grayish areas which in many cases had caseous centers from 2 to 8 mm. in diameter. The interstitial tissue of the lungs was markedly edematous. There was considerable frothy and some purulent exudate in the lumen of the bronchial tree. The intersti- tial tissue of the mediastinum was markedly edematous. The heart was slightly enlarged and dilated and there was considerable interstitial edema of the mediastinum. The posterior mediastinal lymph node was markedly enlarged (50 x 3 x 5 cm.). The architecture of the cortical and medullary regions was obliterated by a yellowish-gray discoloration with streaks and circumscribed foci of cas- eous material. The capsule was thickened. The bronchial lymph nodes were markedly enlarged, one being 3.5 x 7 x 5 cm. and the other 4.3 x l x 3.5 cm. The gross lesions were similar to the above. The anterior mediastinal lymph nodes were markedly en- larged, one being 5 x 3 x 3 cm. and another 3 x 2 x 1 cm. The gross lesions were similar to the above. 86 Histopathologic Findings The lung had multiple circumscribed granulomas with some central caseation necrosis. Many of the lesions were located in the vicinity of the bronchioles as evidenced by remnants of smooth muscle and epithelium within the inflam- matory area. The surrounding alveoli were filled with ser- ous exudate and some alveolar macrOphages. The interstitial tissue was markedly edematous and infiltrated in some areas with inflammatory cells (predominantly neutrophils, lympho- cytes and monocytes). The left ischiatic lymph node had two focal areas of necrosis adjacent to the capsule which were infiltrated with some neutrOphils. The lesions in the left and right bronchial and anter- ior and posterior mediastinal lymph nodes consisted of multi- ple circumscribed foci and confluent areas of caseation necrosis in the vicinity of the subcapsular region which extended by linear streaks into the medullary region. The areas of necrosis were surrounded by a marked granulomatous reaction with giant cells. The capsule was thickened by proliferation of fibrous connective tissue. The liver contained a few scattered foci of epithelioid cells, occasional giant cells and some leukocytic infiltra- tion. There was a moderate fatty metamorphosis of the hepatic cells. 87 Acid-fast bacilli were demonstrated in the lesions of all lymph nodes and organs examined except the left ischiat- ic lymph node and the liver. Bacteriologic Findings Acid-fast bacteria were recovered from (A) the pool of the right and left submaxillary, right and left parotid, right and left medial and lateral retropharyngeal lymph nodes; (B) the pool of the right and left bronchial and anter- ior and posterior mediastinal lymph nodes; (C) the pool of the mesenteric, colic and hepatic lymph nodes; (E) the lung; (H) intestines and (I) the pool of the liver and spleen. Group-III mycobacteria were isolated from one out of ten (l/lO) tubes inoculated from (A), 6/10 tubes from (B), 4/10 tubes from (C), 9/10 tubes from (E), l/lO tubes from (H) and 4/10 tubes from (I). Calf 9O - T. B. Lab. N0. 129 - 63 Clinical History At approximately 21 days after inoculation, clinical signs such as persistent moist cough, partial anorexia and a general loss of body condition were noted. The feed con— sumption decreased to approximately one-third the consump- tion before inoculation. The highest temperature was 106.6 F which was observed 34 days after inoculation. The animal continued to lose weight, and develOped dyspnea (character- ized by abdominal breathing), marked anorexia, serOpurulent 88 nasal discharge, moist rales and a terminal diarrhea. At 47 days after inoculation, the animal was found dead. NecrOpsy Findings (47 days after inoculation) Jersey steer, 7 1/2 months of age, poor condition, weight 250 lbs. The left and right bronchial lymph nodes were markedly enlarged and were 7 x 3.5 x 8 cm. and 6 x 3 x 3.5 cm. One of the bronchial lymph nodes was cut with difficulty and the incised surface had an extensive yellowish-gray discolora- tion with streaks of caseous material extending from the cap- sular region into the medulla. The other bronchial lymph node had similar changes. The anterior mediastinal lymph node was markedly enlarged (10.5 x 2 x 4 cm.) and had gross lesions similar to the above. The posterior mediastinal lymph node was markedly enlarged and was 16 x 3.5 x 7 cm. The lymph node had gross lesions similar to the above. The lungs were markedly enlarged and had patchy areas of red hepatization throughout the parenchyma. The pleura and pulmonary parenchyma had extensive circumscribed foci of solid yellowish-grayish tuberculous tissue some of which con- tained caseous foci from 1 to 3 mm. in diameter. There were more lesions in the apical and cardiac lobes of the lungs than in the diaphragmatic lobes. There was a marked inter- stitial edema in the cardiac and apical lobes. There were 89 reddish fibrinous tags on the visceral and parietal pleura. There was a hydrothorax consisting of approximately 200 ml. of amber colored fluid. The heart appeared to be slightly enlarged and rounded at the apex and there was a marked interstitial edema of the mediastinum. HistOpathologic Findings The lesions in the right and left bronchial and the anterior and posterior mediastinal lymph nodes consisted of large numbers of circumscribed foci and confluent areas of caseation necrosis surrounded by an area of granulomatous reaction with few giant cells. In other sections, the entire lymph node architecture was obliterated by confluent areas of caseation necrosis and granulomas. The hepatic lobules had centrolobular atrOphy and fatty metamorphosis throughout the parenchyma. The pulmonary lesions were characterized by multiple circumscribed foci of granulomatous reaction with central caseation necrosis. The alveoli surrounding the above les- ions contained serous exudate and numerous septal cells and some leukocytes. The lung parenchyma had marked intersti- tial edema. In some areas of the lung the lesions involved bronchioles as evidenced by strands of smooth muscle and epithelium mixed in the inflammatory reaction. Acid-fast bacilli were demonstrated in lesions of all lymph nodes and organs examined. 90 Bacteriologic Findings Acid-fast bacteria were recovered from (A) the pool of the right and left submaxillary, right and left parotid, right and left medial and lateral retropharyngeal lymph nodes; (B) the pool of the right and left bronchial, and anterior and posterior mediastinal lymph nodes; (0) the pool of the mesenteric, colic and hepatic lymph nodes; (E) the lung; (H) the intestines; and (I) the pool of the liver and spleen. Group-III mycobacteria were isolated from 5 out of 10 (5/10) tubes from (A), 8/10 tubes from (B), l/lO tubes from (C), 9/10 tubes from (E), 1/10 tubes from (H), and 4/10 tubes from (I). 2. Calves Exposed to Aerosol with Culture 680-0 Calf 82 - T. B. Lab. N0. 140 - 63 Clinical History On the 2lst day after inoculation the animal had a soft moist cough, partial anorexia and a slight purulent nasal discharge. At approximately 34 days after inoculation the temperature was 104.2 F and it eventually reached 105 F. The persistent, soft moist cough with moist rales, continual loss of weight and partial anorexia resulted in rapid deteriora- tion of the calf’s condition. (Due to anticipated death, the animal was euthanatized and a necropsy was performed. 91 NecrOpsy Findings (51 days after inoculation) Jersey steer, very poor condition, weight 150 lbs. The lungs were markedly enlarged and weighed 4707 gm. Thin slices of lung tissue measuring approximately 1 mm. in thickness were removed from the apical, cardiac and diaphrag- matic lobes of the left and right lung and weighed to the nearest milligram. After fixation, the tubercles present in each slice were counted and an average number of tubercles per gram of lung parenchyma was determined. This was found to be .957 tubercles per gram of lung tissue. 0n the assump- tion that the distribution was the same throughout the lung there were approximately 4500 tubercles present. The lungs had patchy areas of red and gray hepatization over the sur- face and throughout the incised parenchyma. There were large numbers of circumscribed grayish-red and yellowish foci over the incised surface of the lung which were either solid or had caseous centers. These lesions were .1 to 1 cm. in diameter. The posterior mediastinal lymph node was markedly enlarged and was 43 x 12 x 15.5 cm. The node was cut with difficulty and had extensive circumscribed and confluent areas of caseous material obliterating most of the normal architecture of the cortical and medullary regions. The second node was 10 x 3 x 4 cm. and, on incision, similar changes were noted. The capsules were markedly thickened. 92 The bronchial lymph nodes were markedly enlarged ( one was 5.5 x 3 x 4 cm. and the other was 5 x 3 x 3 cm.), with gross lesions similar to the above. The anterior mediastinal lymph nodes were markedly en- larged (one was 7 x 6 x 7 cm. and the other was 5 x 3 x 3 cm.). with gross lesions similar to the above. The liver was friable and had extensive lobular markings and a yellowish discoloration. Histopathologic Findings One of the lateral retrOpharyngeal lymph nodes contained a microscopic focus of epithelioid cells adjacent to the capsule. The right and left parotid lymph nodes had several microscopic foci of epithelioid cells in the peripheral cortical region. There was some infiltration of neutrOphils in and around the focal lesions. The cortical region of the medial retropharyngeal lymph nodes had few microscopic foci of epithelioid cells and neutrOphils. The anterior and posterior mediastinal and right and left bronchial lymph nodes had extensive circumscribed and confluent areas of necrosis obliterating most of the paren- chyma and surrounded by a moderate to extensive granuloma- tous reaction. There was evidence of some early calcifica- tion and leukocytic infiltration. Many of the polymorphonu- clear cells (predominantly neutrophils) were degenerating. The capsules were thickened by fibrous connective tissue. 93 The right prescapular lymph node had one microscopic focus of epithelioid cells in the cortical region. Pulmonary lesions were characterized by circumscribed foci and confluent areas of caseation necrosis surrounded by a marked granulomatous reaction. Many of the lesions appeared to be within the alveolar parenchyma of the lung while others were located in the bronchioles as evidenced by remnants of epithelial or smooth muscle cells. The alveoli surrounding the lesions were filled with a serous exudate, some septal cells and inflammatory cells (neutrOphils and lymphocytes). In other areas the visceral pleura of the lung was raised by a marked fibrous connective tissue proliferation in which foci of granulomatous reaction were found. There were very few giant cells in the inflammatory area. N There was fatty metamorphosis of the distal convoluted tubules of the kidneys. The liver had a marked fatty metamorphosis and early centrolobular coagulation necrosis. One tissue section of the internal iliac lymph node contained a micros00pic focus of epithelioid cells. The right and left deep inguinal lymph nodes contained microscopic foci of epithelioid cells in the cortical region. Acid-fast bacilli were demonstrated in lesions of all the lymph nodes and organs examined except the right and left deep inguinal and medial retrOpharyngeal lymph nodes. 94 Bacteriologic Findings Acid-fast bacteria were recovered from (A) the pool of the right and left submaxillary, right and left parotid, right and left medial and lateral retrOpharyngeal lymph nodes; (E) the lung and (I) the pool of the liver and spleen. Group-III mycobacteria were isolated from three out of 10 tubes (3/10) from (A), 9/10 tubes from (E) and 8/10 tubes from (I). Calf 86 - T. B. Lab. No. 173 - 63 Clinical History The calf had a soft, moist, persistent cough, rales and some loss of body condition starting at approximately 21 days after inoculation. Thirty-five days after inoculation, there was in addition to the above a seropurulent nasal discharge. The rectal temperature of the animal varied throughout the experiment, reaching a high of 103.8 F on the 35th day after inoculation. The animal continued to lose condition and a fetid diarrhea was noticed starting approximately 65 days after inoculation. The animal steadily deteriorated and when death became imminent, euthanasia and necrOpsy were performed. NecrOpsy Findings (76 days after inoculation) Jersey steer, approximately 9 months of age, poor condition, body weight approximately 250 lbs. The bronchial lymph nodes were markedly enlarged. One was 7 x 4 x 4.5 cm. and the other 8 x 7 x 7 cm. The lymph 95 nodes were cut with difficulty and the incised surface had wideSpread circumscribed foci and confluent areas of yellow- ish-gray discoloration and caseous material obliterating most of the normal architecture. The capsules of the lymph nodes were thickened. The anterior mediastinal lymph nodes were markedly enlarged. The first was 5.5 x 4 x 7 cm., the second 6 x 4 x 4 cm., and the third 5.5 x 4 x 4.5 cm. The lymph nodes had gross lesions similar to the above. The posterior mediastinal lymph nodes were markedly enlarged (Fig. 12). The first was 20 x 6.5 x 8 cm., the second 5 x 3.5 x 4 cm., the third 7 x 4 x 5 cm. and the fourth 5 x 3.5 x 2.5 cm. The lymph nodes had gross lesions similar to the above. Several mesenteric lymph nodes had irregularly shaped and circular areas of yellowish caseous material 2 to 5 mm. in diameter. The lungs were enlarged and had patchy areas of red to gray hepatization in all lobes. Many 2-to-3-cm. raised and irregularly shaped to circular caseous areas were found in the pleura. The lung parenchyma was cut with some diffi- culty and had large numbers of circumscribed foci of caseous material 2 to 30 mm. in diameter. The bronchi contained serOpurulent exudate. There was marked interstitial edema involving all lobes of the lungs. Approximately 50 ml. of serosanguineous fluid were pre- sent in the thoracic cavity. There was marked interstitial Fig. 12. Posterior mediastinal lymph node from calf 86, 76 days after exposure to culture 68C-0. of the lymph node. Note the size 96 97 edema of mediastinal tissue particularly in the vicinity of the heart. The heart was slightly enlarged and rounded at the apex. There was a circumscribed focus of yellowish dis- coloration approximately 4 cm. in diameter in the left coro- nary groove, approximately 2.5 cm. from the apex of the heart, in the left ventricle. Scattered throughout the Splenic parenchyma were several circumscribed yellowish foci from 2 to 4 mm. in diameter. The liver had several yellowish, circumscribed foci (l to 5 mm. in diameter), some of which had caseous material, scattered throughout the organ. One of the hepatic lymph nodes had a few circumscribed yellowish foci of caseous material. The right and left kidneys had several scattered caseous foci, from 2 to 3 mm. in diameter. There were a few large, yellowish, triangular-shaped lesions in the cortex, the apex of which extended to the cortico-medullary junction. Histopathologic Findings The pulmonary lesions consisted of several circumscribed foci of caseation necrosis and calcification surrounded by a marked granulomatous reaction. In some of the lesions there was also a marked lymphocytic infiltration. The alveoli surrounding the lesions and some of the bronchioles were filled with neutrophils. Extending from and confluent with the more advanced caseous lesions were non-caseating focal granulomas. 98 The hepatic lesions consisted of several circumscribed foci of granulomas, some of which had central caseation ne- crosis. There was diffuse fatty metamorphosis of the liver parenchyma. The subcapsular sinus of the left ischiatic lymph node in some areas was partially filled with inflammatory cells consisting of macrOphages and neutrophils. The lesions were suggestive of embolic origin. There was a circumscribed microscopic focus of granulo- matous reaction and leukocytic infiltration in the left pop- liteal lymph node. The left and right medial retropharyngeal lymph nodes had multiple circumscribed foci and confluent areas of non- caseating and caseating granulomatous tissue, in the cortex adjacent to the capsule. The lesions also contained a mod- erate leukocytic infiltration. The submaxillary lymph nodes had few circumscribed micros00pic foci of granulomatous reaction and leukocytic infiltration. The lesions in the right and left bronchial and the anterior and posterior mediastinal lymph nodes consisted of diffuse coagulation and caseation necrosis obliterating most of the lymph node parenchyma and surrounded by a narrow zone of granulomatous reaction. There were streaks of calcifica- tion, areas of edema and leukocytic infiltration within the areas of necrosis. No evidence of giant cell formation was 99 present. The capsule was thickened by fibrous connective tissue. The spleen had several large foci of caseation necrosis surrounded by epithelioid cells and neutrOphils. The latter cells also extended into the necrotic areas. The lesions appeared to have their origin within the white pulp. The gastric lymph nodes were characterized by multiple circumscribed and confluent areas of caseation necrosis sur- rounded by a granulomatous reaction. The mesenteric lymph nodes had multiple circumscribed and confluent foci of caseating and non-caseating granulomas. The cortical region of the hepatic lymph node had many circumscribed foci of granulomatous reaction, some with caseation necrosis. There was also a moderate leukocytic infiltration. The left prescapular lymph node had a few microscopic foci of epithelioid cells scattered throughout the cortex of the node. The right and left prefemoral lymph nodes had few micro- scopic foci of granulomas in the cortical region. The liver had several microscopic caseating and non- caseating granulomas. A few giant cells were seen in these areas and there was some lymphocytic infiltration into and around the granulomas. There was moderate fatty metamorpho- sis of the liver parenchyma. The heart lesion was characterized by a focal area of degenerative changes in the muscle which included increase 100 of eosinophilic staining, loss of striations, cloudy swelling and in some areas early calcification. The nuclei of many of the muscle cells were pyknotic. In some areas, there was an increase of fibrous connective tissue. Numerous heart myo- cytes (Anitschkow's cells) were observed in the degenerated area of the heart muscle. The cortical region of the superficial inguinal lymph nodes had few circumscribed foci of granulomatous reaction. In one of the foci, giant cells were present. The right ischiatic lymph node lesions had few circum- scribed granulomas with central caseation necrosis and some leukocytic infiltration. The renal lesions consisted of circumscribed granulomas with giant cells and central caseation. There were also large areas of tubular necrosis and early calcification. Acid-fast bacilli were demonstrated in lesions of all lymph nodes and organs examined except the right and left superficial inguinal and left ischiatic lymph nodes. Bacteriologic Findings Acid-fast organisms were recovered from (A) the pool of the right and left submaxillary, right and left parotid, right and left medial and lateral retropharyngeal lymph nodes; (B) the pool of the right and left bronchial and the anterior and posterior mediastinal lymph nodes; (0) the pool of the mesenteric, colic and hepatic lymph nodes; (E) the lung; (H) the intestines; and (I) the pool of the liver and Spleen. 101 Group-III mycobacteria were isolated from nine out of 10 tubes (9/10) from (A), 3/10 tubes from (B), 1/10 tubes from (C), 4/10 tubes from (E), 2/10 tubes from (H) and 1/10 tubes from (I). Calf 83 - T. B. Lab. N0. 172 - 63 Clinical History At approximately 22 days after inoculation the animal had signs of partial anorexia, a persistent, soft moist cough, and a serous nasal discharge. A rectal temperature of 105 F was noted 35 days after inoculation, but it returned to nor- mal in the terminal stages of the disease. There was consid- erable loss of body weight starting approximately 21 days after inoculation. A moderate depression and diarrhea started approximately 64 days after inoculation. Due to impending death the animal was euthanatized and necropsy performed. Necropsy Findings (78 days after inoculation) Jersey steer, 8 months old, poor condition, weight approxi- mately 150 lbs. The adipose tissue of much of the carcass, particularly around the base of the heart, was gelatinous in appearance. Three lymph nodes of the hepatic group were slightly enlarged and had multiple circumscribed foci of caseous material from 3 to 5 mm. in diameter. The liver was slightly enlarged, rounded at the free edges and contained a number of circumscribed foci of caseous 102 material 2 to 8 mm. in diameter. There were also circum- scribed yellowish foci (approximately 1 x 1.5 x 1 cm.) in the liver which extended from the capsule into the parenchy- ma and resembled infarction. The liver had prominent lobular markings and a yellow discoloration. Both kidneys had scattered, circumscribed, yellowish, caseous foci from 2 to 4 mm. in diameter in the cortical and medullary regions (Fig. 18). There were also multiple, well demarcated lesions, resembling infarction, in the cortex consisting of yellowish to greenish cone-shaped foci. The apex of the lesions started at the cortico—medullary junc- tion and at the surface were 1.5 to 2 cm. in diameter. The parietal and visceral pleura supported numerous, scattered, small, reddish colored, fibrinous plaques over the surface. The heart was moderately enlarged and rounded at the apex. The epicardial adipose tissue was gelatinous in appearance. The lungs were markedly enlarged and had scattered, patchy areas of red to gray hepatization over the surface of all lobes. There were numerous circular and irregularly shaped foci of yellow caseous material varying from .2 to 2.5 cm. in diameter (Fig. 13). The lesions were uniformly distributed throughout all lobes. Some of the larger lesions were encapsulated. One lymph node of the mesenteric group had two circum- scribed foci of caseous material from 3 to 5 mm. in diameter. Fig. 13. Lung from calf 83, 78 days after aerosol exposure with culture 680-0. Note extensive circum- scribed foci and confluence of tuberculous lesions. 103 104 One lymph node of the colic group had a few circum- scribed foci of caseous material 1 to 2 mm. in diameter. Lymph nodes of the anterior mediastinal group were markedly enlarged; the first lymph node was 8 x 4.5 x 5 cm., the second, 4.5 x 3.5 x 4 cm. and the third, 7 x 2.5 x 4.5 cm. The lymph nodes were cut with difficulty and the cut surface had extensive circumscribed foci and confluent areas of caseous material obliterating most of the parenchyma. The posterior mediastinal lymph nodes were also marked- ly enlarged; the first lymph node was 15.5 x 5 x 7.5 cm., the second lymph node 5 x 3 x 2.5 cm., and the third lymph node 4 x 3 x 2 cm. The lymph nodes had gross lesions similar to the above (Fig. 14). The right and left bronchial lymph nodes were markedly enlarged, one being 8 x 3 x 5.5 cm., and the other 10 x 5 x 7 cm. The lymph nodes had gross lesions similar to the above. HistOpathologic Findings The cortical region of the right and left medial retro- pharyngeal lymph nodes had three microscopic foci of granulo- matous reaction and neutrOphilic infiltration. One of the right and left parotid lymph nodes had a small circumscribed focus of granulomatous reaction with early central caseation necrosis. The lesion was infil- trated with neutrophils. Adjacent to this lesion, there were foci composed of solid clusters of epithelioid cells. 105 Fig. 14. Posterior mediastinal lymph node from calf 83, 78 days after exposure to culture 680-0. The normal architecture was obliterated by necrotic tissue. Fig. 15. Posterior mediastinal lymph node from calf 83,78 days after aerosol exposure with culture 680-0. Note area of caseous granuloma. New Fuchsin--H & E. x75. 106 The colic lymph node had several circumscribed foci of caseation necrosis surrounded by zones of granulomatous reac- tion with some neutrOphilic infiltration. Extending from these lesions were several solid clusters of epithelioid cells. The gastric lymph node had a few circumscribed foci of caseation necrosis surrounded by a granulomatous reaction. In other parts of the node noncaseating granulomas were found. The mesenteric lymph node had several irregularly shaped and rounded foci of caseation necrosis surrounded by a granu- lomatous reaction and neutrOphilic infiltration. The pulmonary lesions were characterized by multiple circumscribed foci and confluent areas in which there was granulomatous tissue with central caseation necrosis. The lesions were located in bronchioles, alveoli, and, in some cases, the interstitial tissue. Many of the lesions were surrounded by infiltrates of lymphoid cells. In other areas the alveoli around the lesions were filled with macrophages, some neutrophils and serous fluid. The lung parenchyma had areas of atelectasis and emphysema. There was a marked interstitial edema in which lymphocytic infiltration and fibroblastic proliferation occurred. The liver had a large circumscribed focus of caseation necrosis surrounded by a narrow zone of epithelioid cells and lymphocytes. The parenchymal cells of the liver had undergone cloudy swelling and early fatty metamorphosis. 107 Throughout the parenchyma were micrOSCOpic foci of monocytic and lymphocytic infiltration. The spleen had a microsc0pic focus of coagulation necro- sis in which there was an infiltration of neutrOphilic cells and a few epithelioid cells. The right popliteal lymph node had a small circumscribed focus of caseation necrosis surrounded by a narrow zone of epithelioid cells. Only an occasional giant cell could be found in the inflammatory reaction. The left ischiatic lymph node had a circumscribed focus of caseation necrosis surrounded by a narrow zone of epithe- lioid and some neutrophilic cells. An occasional giant cell was noted. The right prefemoral lymph node had two cortical foci of granulomatous reaction. Both of these lesions had early central caseation necrosis. The left axillary lymph node had a circumscribed focus of caseation necrosis surrounded by a narrow zone of epithe- lioid cells, neutrophils and occasional giant cells. The architecture of the anterior and posterior medi- astinal and right and left bronchial lymph nodes was almost obliterated by circumscribed and confluent areas of caseation necrosis surrounded by narrow zones of epithelioid cells which themselves had undergone considerable necrosis (Figs. 15, 16 & 17). The inflammatory area also contained some neutrOphils and lymphocytes. There was evidence of scattered areas of early calcification throughout the caseous centers. 108 Fig. 16. A different area of the same lymph node as in Fig. 15. Note the extensive cellular necrosis of the zone bordering the area of caseation necrosis. New Fuchsin--H & E. x187. Fig. 17. Higher magnification of zone of cellular necrosis shown in Fig. 16. There is extensive pyknosis and karyorrhexis. New Fuchsin--H & E. x400. 109 Fig. 18. Kidney from calf 83, 78 days after aerosol exposure to culture 680-0. Note infarcted area (1) and several subcapsular tuberculous lesions (arrows). . .; -23 ... Fig. 19. Kidney from calf 83, 78 days after aerosol exposure to culture 680-0. There is a large area of casea- tion necrosis surrounded by a zone of granulomatous reaction. New Fuchsin--H & E. x75. 1. L‘ . 110 The capsules of the lymph nodes were thickened by fibrous connective tissue. Occasionally a giant cell was noted in the inflammatory areas. The hepatic lymph node had several irregularly shaped to circular foci of caseation necrosis surrounded by a zone of epithelioid cells with some neutrOphils and early calci- fication in the caseous centers. The subcapsular renal lesions consisted of circumscribed foci of caseation necrosis surrounded by narrow zones of granu- lomatous reaction (Fig. 19). In other parts of the cortex, there were wide areas of tubular necrosis and calcification (Figs. 20 & 21). Acid-fast bacilli were demonstrated in the lesions of all lymph nodes and organs except the right p0pliteal and prefemoral lymph nodes and spleen. Bacteriologic Findings Acid-fast organisms were recovered from (A) the pool of the right and left submaxillary, right and left parotid, right and left medial and lateral retropharyngeal lymph nodes; (B) the pool of the right and left bronchial, and the anter- ior and posterior mediastinal lymph nodes; (0) the pool of the mesenteric, colic and hepatic lymph nodes, (E) the lung; (H) the intestine; and (I) the pool of the liver and Spleen. Group-III mycobacteria were isolated from ten out of 10 tubes (10/10) from (A), 5/10 tubes from (B), 2/10 tubes from (o), 10/10 tubes from (E), 1/1o tubes from (H) and 2/10 tubes from (I). 111 Fig. 20. Kidney from calf 83, 78 days after aerosol exposure with culture 680-0. Note the extensive tubular degeneration and calcification. New Fuchsin--H & E. x75. Fig. 21. Higher magnification of tubules in Fig. 20. There is a marked coagulative necrosis and karyolytic loss of nuclei of the renal tubules. New Fuchsin--H & E. x187. 112 3. Calves Exposed to Aerosol with Culture 50B~0 Calf 89 - T. B. Lab. No. 146 - 63 Clinical History The animal had no clinical signs of disease throughout the course of the experiment. The rectal temperature remained normal. Necropsy Findings (64 days after inoculation) Jersey steer, 8 months old, excellent condition, weight 450 lbs. There were no detectable gross lesions in any of the lymph nodes or organs examined. HistOpathologic Findings The liver had a few circumscribed foci of necrosis surrounded by an eosinOphilic infiltration. Acid-fast bacilli were not demonstrated. There were no detectable microscopic lesions in the remaining organs and lymph nodes. Acid-fast bacilli were not demonstrated. Acid-fast bacteria were recovered from (A) the pool of the right and left submaxillary, right and left parotid, right and left medial and lateral retropharyngeal lymph nodes; (B) the pool of the right and left bronchial, and the anterior and posterior mediastinal lymph nodes; (0) the pool of the mesenteric, colic and hepatic lymph nodes, (E) the lung; and (I) the pool of the liver and spleen. ‘ 113 Group-III mycobacteria were isolated from one out of 10 (1/10) tubes from (A), 3/10 tubes from (B), 1/10 tubes from (C), 4/10 tubes from (E) and 1/10 tubes from (I). Calf 87 - T. B. Lab. NO. 228 - 63 Clinical History There were no signs of disease throughout the course of the experiment. Necropsy Findings (128 days after inoculation) Jersey steer, weight approximately 600 lbs, excellent physi- cal condition. There were no detectable gross lesions in any of the organs or lymph nodes examined. HistOpathologic Findings There were no detectable microscopic lesions in any of the organs or lymph nodes examined. Acid-fast bacilli were not demonstrated. Bacteriologic Findings Acid-fast bacteria were recovered from (B) the pool of the right and left bronchial and anterior and posterior mediastinal lymph nodes. Group—III mycobacteria were isolated from all ten tubes of culture media used (10/10). 114 Calf 88 - T. B. Lab. NO. 294 - 63 Clinical History The calf had no signs of overt disease during the course of the experiment. NecrOpsy Findings (188 days after inoculation) Jersey steer, excellent condition, weight approximately 450 lbs, approximately 12 months of age. The cut surface of some of the mesenteric lymph nodes exuded a white fluid resembling chyme. No other gross lesions were noted in the animal. Histopathologic Findings No microscopic lesions were found in any of the organs or lymph nodes examined. Acid-fast bacilli were not demon- strated. Bacteriologic Findings Acid-fast bacteria were recovered from (B) the pool of the right and left bronchial and the anterior and posterior mediastinal lymph nodes. Group-III mycobacteria were recov- ered from seven of the ten tubes of culture media used (7/10). 115 C. Tuberculin Sensitivity Studies Tables 3, 4 and 5 contain the tuberculin test results for the intrauterine experiment. All test measurements were recorded in millimeters and corrected by subtraction of the zero hour skin thickness from skin thickness at 24, 48 and 72 hours after injection of the tuberculin. Tables 6, 7 and 8 contain the tuberculin test results of the aerosol experiment. 116 . omen» muons—ous... 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