mam m swms RESULTING mom EXPERIMENTAL WENB‘ERATEON OF MYCOBACTERMM BOVIS. Mycommmm AVIUM. m GROUP-m MW Thesis {or the Dames of Ph. D. MICHIGAN flA‘i’E UNIVER‘SM’ James A, Ray 1966 Jt‘lhblb This is to certify that the thesis entitled DISEASE IN SWINE RESULTING FROM EXPERIMENI‘AL ADMINISTRATION OF MYCOBACTERIUM BOVIS, MYCOBACI‘ERIUM AVIUM. 0R GROUP-III MYCOBACTERIA presented by James A. Ray has been accepted towards fulfillment of the requirements for Ph.D. degree in Pgthglogy x' "}?i’,." "f (1/. C fl /,///<:1/4L,1L£ (é/3 Major farofessor Date ._J£_1_Y_15+_19.6g__ 0—169 ABSTRACT DISEASE IN SWINE RESULTING FROM EXPERIMENTAL ADMINISTRATION or MYCOBACTERIIM s_oy_1_s.. , MYCOBACTERIUM w, OR GROUP-III MYCOBACTERIA by James A. Ray To each of 39 Yorkshire-Berkshire-Iandrace crossbred pigs, approxi- mately 8 to 11 weeks old, 2 mg. (wet weight) of cells of l of 9 myco- bacterial strains were administered orally or by intradermal injection of the left foreleg. The pigs were housed in isolation by groups 1) according to the route of administration and 2) so as to prevent cross- infection. Twenty-one additional pigs (litter-mates) were housed with the above-mentioned pigs to detect transmission by contact. Fifteen other litter-mates were housed in groups under similar conditions to constitute uninoculated controls. The strains of organisms administered were as follows: 1 yycoba - £33.92 1331.; of. porcine origin; 4 Group-III mycobacteria of porcine origin; 2 Group-III mycobacteria of pen origin; 1 g. 23.21.! of porcine origin; and l g. M, laboratory strain. The pigs were observed for signs of disease and tested with both avian and mamalian tuberculins during the course of the experiment. At various intervals during the experiment pigs were selected arbitrarily and killed. Pathologic and bacteriologic examinations were conducted on the tissues. James A. Ray No signs of disease other than swelling or swelling and ulceration of the intradermal inoculation site were observed. In general, the lesions deve10ping at the injection site were in decreasing order of severity following injection of 1!. m, Group-III mycobacteria of porcine origin, 1:1,. Ell-£9. of porcine origin and y. m, laboratory strain. Almost no change was produced by Group-III mycobacteria of pen origin. Following ulceration, which occurred in most cases where lesions develOped, the lesions appeared to heal. All pigs to which y, 2011.3 was administered deve10ped greater response to mamalian tuberculin than to avian tuberculin. When 3. $23.29 was ad- ministered, the responses to avian tuberculin exceeded those to mammalian tuberculin except in 2 pigs tested 62 days after inoculation. One was killed shortly after this test; the other responded in greater degree to avian tuberculin on a subsequent test. All pigs receiving Group-III mycobacteria of porcine origin had greater response to avian tuberculin than to mammalian tuberculin. There was only 1 measurable response (1 mm. swelling to avian tuberculin) in the pigs receiving Group-III mycobacteria of pen origin. Bacteriologic data indicate that acid-fast organisms were widespread in all pigs except those inoculated with Group-III mycobacteria of pen origin and the uninoculated control pigs. With only 4 exceptions, acid- fast organisms were isolated from all tissues where lesions were found. In 83 pools of tissue out of 280 when no lesions were found, acid-fast organisms were isolated. In no instance was a mycobacterium isolated from.a noncontact uninoculated control pig. Lesions were found in most animals receiving y. m, either 31. gvium strain, or any of the Group-III mycobacteria of porcine origin and James A. Ray each strain produced lesions in at least 1 pig when administered by either route. None were found when Group-III mycobacteria of pen origin were injected. The Group-III mycobacteria of porcine origin produced more extensive lesions than did :1, M and less extensive lesions than did g. 2933. In certain instances, the extent of disease caused by '15,. 311.2 or Group-III mycobacteria of porcine origin appeared to lessen with the passage of time. The lesions caused by £1. a_vi_u_n;, 31. Egg}; or Group-III mycobacteria of porcine origin could not be differentiated macroscopically or microscopically. lesions were found in the tissues of the pen-mates of pigs receiving 11. fill—‘9.“ (laboratory strain) intradermally, y. m (porcine origin) orally, :4. mg; orally, or Group-III mycobacteria (porcine origin) orally. (There were no pen-mates with those receiving 31. m intra- dermally.) Acid-fast organisms were isolated from all pen-mates except 1 killed 52 days after :3. m, laboratory strain, was administered orally to other pigs housed with it. I No tuberculin hypersensitivity, lesions or acid-fast organisms were found in the uninoculated control pigs. DISEASE IN SWINE RESUETING FROM EXPERIMENTAL ADMINISTRAle OF MYCOBACTERIUM BOVIS, MYCOBACTERIUM AVIUM, OR GROUP-III MYCOBACTERIA By CV~ James AyfRay A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pathology 1966 To Nancy, Tansy and Carolyn ACKNW IEDGEMENIS This study was conducted as a cooperative project of the Departments of Microbiology and Public Health, Pathology, and Surgery and Medicine of Michigan State University and the U. S. Department of Agriculture. It is the result of the work of many persons. The author is grateful to all who made this study possible. Following are the names of those to whom the author is particularly indebted for their direct assistance in planning and conducting the study and preparing this manuscript. Dr. D. Dr. R. J. Ellis, Department of Surgery and Medicine Goyings, Department of Pathology, Mallmann, Department of Microbiology and Public Health Mallmann, Department of Microbiology and Public Health McGavin, Department of Pathology Morrill, Department of Pathology Scott, U. S. Department of Agriculture In addition, the willing assistance and cooperation of the staff of the Tuberculosis Research Project and the Pathology Department are gratefully acknowledged . ii TABLE INTRODIETICN........ REVIEW OF LITERATURE. . . . MATERIALS AND METHODS . . . Experimental Animals . Cultures....... OF CONTENTS Administration of Mycobacteria Clinical Examinations. . . . . Pathologie Techniques. . . Bacteriologic Techniques . Tuberculin Test Techniques Hematologic Techniques . . mums O C C O O O O O O O O O A. Intradermal Inoculation. . . Culture 81-0, !. bovis, swine origin. Culture 167C1-1, Group III, swine origin. Culture 1860-1, Group III, swine origin . Culture 172C1-1, Group III, swine origin. Culture 1932-1, Group III, swine origin . fl. avitnn, laboratory strain . . . . . . . Culture 206-2, 94. gins, swine origin . . Culture 15D, Group III, pen origin. . . . Culture 122W, Group III, pen origin . . . iii Page 17 17 l7 l9 19 20 21 22 23 28 29 29 37 45 47 49 54 62 63 Page B. Oral Administration. . . . . . . . . . . . . . . . . . . . . 64 Culture 81-0, gm, swine origin. . . . . . . . . . 64 Culture 172C2-l, Group III, swine origin. . . . . . . . 71 §.gli_._t_1_m,1aboratorystrain....l.......... 75 Culture 206-1, gm, swine origin. . . . . . . . . 77 C. uninoculated Control Animals . . . . . . . . . . . . . . . . 81 DISCUSSION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 Clinical Findings. . . . . . . . . . . . . . . . . . . . . . . . 103 Pathologic Findings. . . . . . . . . . . . . . . . . . . . . . . 105 Characteristics of the lesions. . . . . . . . . . . . . 105 Extent of Disease . . . . . . . . . . . . . . . . . . . 110 Bacteriologic Findings . . . . . . . . . . . . . . . . . . . . . 118 Tuberculin Tests . . . . . . . . . . . . . . . . . . . . . . . . 120 The Possible Resolution of Lesions . . . . . . . . . . . . . . . 123 SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 LISTWREFEREICES..........................128 VITA. O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 136 iv Table LIST OF TABLES Page Index of pig number, housing schedule, route of adminis- tration, culture number and source, culture classifica- tion, duration of experiment, and page number in results. . 24 Summary of lesions and bacteriologic isolations from pigs t0‘which mycobacteria were administered . . . . . . . . . . 83 Increase in dermal thickness 48 hours after injection of avian and mammalian tuberculins at various intervals following exposure to selected mycobacteria . . . . . . . . 94 Results of hematologic examinations of blood samples taken immediately prior to necropsy . . . . . . . . . . . . . . . 100 Figure 10 ll 12 13 LIST (I FIGURES Left prescapular lymph node of Fig 2-2, inoculated intradermally with Culture 81-0, M. bovis, swine origin . Left prescapular lymph node of Pig 2-2, inoculated intradermally with Culture 81-0, M. bovis, swine origin . Spleen of Pig 2-1, inoculated intradermally with Culture 81'0,§.b0v18,8w1neorlgln. e e e e e e e e e e e e e e Lung of Pig 2-1, inoculated intradermally with Culture 81-0, M. bovis, swine origin. . . . . . . . . . . . . . . lung of Pig 2-1, inoculated intradermally with Culture 81-0, E. bOVia’ Wine origin. 0 O O O O O O O O O O O O 0 left prescapular lymph node of Pig 2-4, inoculated intra- dermally with Culture 81-0, M. bovis, swine origin. . . . Liver of Pig 2-4 inoculated intradermally with Culture 81-0,M.bovis, swineorigin. . . . . . . . . . . . . . . Left prescapular lymph node of Pig 3-4, inoculated intra- dermally with Culture 16701-1, Group-III mycobacteria, Wine origin. 0 O O O O O O O O O I O O O O O O O O C O 0 Left prescapular lymph node of Pig 3-4, inoculated intra- dermally with Culture 167C1-l, Group-III mycobacteria, sw1ne°rigineeeeeeeeeeeeeeeeeeeeeee Right bronchial lymph node of Pig 3-4, inoculated intra- dermally with Culture 167C -1, Group-III mycobacteria, 1 wine origin. 0 O O O O O O O O O O O O O O I O O O O O 0 Left prescapular lymph node of Pig 5-3, inoculated intra- dermally with M. avium, laboratory strain . . . . . . . . Mesenteric lymph node of Pig 5-6, inoculated intradermally with M. avium, laboratory strain. . . . . . . . . . . . . . Lateral retropharyngeal lymph node of Pig 10-1, inoculated intradermally with Culture 206-2, M. avium, swine origin. . vi' Page 31 31 34 36 36 38 38 4O 40 41 51 55 Figure 16 Page Lateral retrOpharyngeal lymph node of Pig lO-l, inoculated intradermally with Culture 206-2, M. gvium, swine origin. . 58 Left submaxillary lymph node of Fig 6-3, to which Culture 1720 -l, Group-III mycobacteria, swine origin was adm£nistered orally 0 O O O O O O O O O ,0 O O O O O O O O O 75 Mesenteric lymph node of Pig 6-3, to which Culture 172C1-1, Group-III mycobacteria, swine origin was administered orally. 0 O O O O O O O O I O O O O O O O O O O O O O O O O 75 vii INTRODUCT ION Probably no affliction has caused more death, suffering and economic loss throughout the years than tuberculosis. It is undoubtedly the most studied of all bacterial diseases. Yet with all this study tuberculosis is not fully understood and is still the greatest killer of man in the world (Smith;e_t_ 31., 1964). Even in the United States where it is well controlled it remains the most prevalent microbial cause of death (Dubos, 1954). The incidence of tuberculosis in man has drapped markedly in the United States during the 20th century. This is probably due to improved living conditions and sanitation as well as to an effective control pro- gram. This program was designed to step the spread of theorganism by the detection, isolation and treatment of persons shedding tubercle bacilli. Because of its economic and public health importance, bovine tubercu- losis has been subjected to extensive and relatively effective control programs. These programs have greatly reduced the incidence of the disease but have not eliminated it. No such control program has been applied to porcine tuberculosis. This is true in spite of substantial economic losses and a potential, if not real, public health hazard afforded by this disease. While the infec- tion rate in the United States as indicated by meat inspection reports 2 appears low (1.42%) (U. S. Department of Agriculture, 1965) it is 855 times that found in cattle during the same period. Since swine can be affected by Mycobacterium tuberculosis, M. Mg, or M. 211.22: tubercu- lous pigs are potential hazards for man, cattle, or fowl as well as other pigs. When Group-III mycobacteria were isolated from tissues of more than 100 pigs (Mallmann, Mallmann and Ray, 1962), the question of the impor- tance of pigs in the transmission of this disease was raised. Similar organism had been incriminated as the source of disease in human beings (Runyon, 1959) and cattle (Mallmann, 1961). Epidemiologic studies of such cases in human beings indicated that the disease was not spread from man to man (Edwards and Palmer, 1959). While no positive source was found, Edwards and Palmer (1959) had found high rates of reaction (40%, 15%, 151, respectively, of persons fran 3 of 5 farms to a purified-protein- derived (PPD) tuberculin made from a Group-III mycobacterium of human origin. Subsequently, McGavin (1964) was able to produce a primary complex (lesions at intradermal inoculation site and lymph node draining it) in only 1 of 11 calves when each of these animals was inoculated intradermally with 1.0 mg. (wet weight) of l or 6 Group-III mycobacteria of swine origin isolated by Maan 3; .a_1_. (1962). In general, the Group-III mycobacteria used in his study were less virulent for calves than were the Group-III mycobacteria of bovine origin. The purposes of this study were 1) to reproduce the disease in swine with Group-III mycobacteria of swine origin and to reisolate the organisms from the tissues of the infected animals, thereby fulfilling Koch's 3 postulates, 2) determine if transmission occurred from pig to pig, and 3) compare the disease caused by these organisms to that produced by M. m and M. 22.1.29. under similar conditions. The organisms were administered intradermally and orally as these routes seemed the most likely under natural conditions. REVIEW OF LITERATURE Since before recorded history, tuberculosis has afflicted man. Lesions resembling those of tuberculosis have been found in the bones of Egyptian mummies (Webb, 1936). Tuberculosis has killed more men than any other affliction. It is still the most common cause of death in man (Smith 2; g_1_., 1964). Many species of animal, including many birds and cold-blooded animals, have been found to be tuberculous (Francis, 1958). Tuberculosis of cattle, swine and birds is the most important from the economic and public health standpoint. Bovine tuberculosis has been reduced in the United States by a rigorous eradication program. The incidence of tuberculin reactors was reduced from 52 in 1917 to 0.11% in 1954 (Jehnson, Baisden, and Frank, 1961). However, Mallmann, Mallmann and Ray (1964) noted that the inci- dence of bovine tuberculosis has changed little since 1940. Hence the program has become a control program instead of an eradication pro- gram. Even as a control program, it is of great value. In Europe, when inadequate control methods were used, the incidence of tuberculosis was about 352 in cattle 5 years old (Francis, 1958). The incidence of avian tuberculosis has been greatly reduced by the increased practice of having only pullet flocks. Schalk e§_gl. (1935) found pathogenic M. 2.11:2! in chicken litter 4 years after it was voided. They felt that disease could not be controlled by the slaughter of 5 tuberculin-positive birds because of soil contamination, and recommended eliminating all birds more than 1 year old from the flocks. Lee (1955) found 0.241 tuberculin-positive chickens when testing 33,769 birds from all-pullet flocks, while 11.521 of 5,382 hens 2 or more years old were tuberculin-positive. Pickard (1952) reported that 14% of 119 pullet flocks tested contained tuberculin-positive birds, while 572 of 95 hen flocks tested contained birds that were positive. Therefore, much of the tuberculosis in chickens can be eliminated by having only all-pullet flocks. Following an extensive review of European and American literature, Francis (1958) stated that the incidence of porcine tuberculosis varied from 1 to 52 early in the 20th century. In the United States, the inci- dence was 22 in 1908 (Francis, 1958). It increased to a high 16.38% in 1922 (Karlson, 1964). This was thought to be due to M. £332 infections transmitted to the pig from tuberculous poultry. The incidence of fowl tuberculosis decreased markedly with the change to all-pullet flocks (Francis, 1958; Karlson, 1964). During the same period the incidence of porcine tuberculosis dropped, reaching a level of 1.422 in 1965 (U. S. Department of Agriculture, 1965). Bacteriologic studies of 23 workers, summarized by Francis (1958), indicate that porcine tuberculosis was caused by M. tuberculosis, M. 13211.; and M. 932. From this review and a similar review by [Carlson (1964), it is clear that M. 91.1.9.9 is the most comon isolant of the 3 afore- mentioned species. Griffith established that subcutaneous inoculations of M. M911; (1907), M. 93.1.2! and M. tuberculosis (1911) could cause tuberculosis in swine. 6 Porcine tuberculosis is mainly associated with the exposure of pigs to products, excreta, or diseased tissue of tuberculous cattle, fowls or human beings. Mahler and Washburn (1908 and 1917), Thornton (1961) and Anthony (1950) reported finding higher incidences of tuberculosis in pigs from areas where bovine tuberculosis existed. Dairy products, returned to farms to be used as swine feed, were recognized early as a source of infection. This is evidenced by early laws requiring the pasteurization of dairy products used as animal feeds in Denmark (Bang, 1908) and the United States (Mahler and Washburn, 1908). The latter preceded the requirement of pasteurization of dairy products for human use. Mycobacterium.tuberculosis has been isolated from tuberculous lesions of swine from EurOpe (Francis, 1958), the Philippines (Topacio, 1933), South Africa CRobinson, 1958), Japan (Hatokeyama 25.31,, 1961) and the United States (Feldman, 1939). In all instances, the pigs had had direct or indirect contact with tuberculous human beings. While earlwaorkers had shown that porcine tuberculosis could be caused by M. Ell-.9 (Weber and Bofinger, 1907; Mohler and Washburn, 1908; Griffith, 1911), it was thought that most of it was due to M. 2211.; and that the control of bovine tuberculosis would also control porcine tuberculosis (Mahler and Washburn, 1908). When the incidence of tubercu- losis in swine increased after the start of the bovine tuberculosis eradication project (Feldman, 1938; Karlson, 1964), attempts were made to determine the cause. 7 Van Es and Martin (1925) found M. Ell—‘2 in 74.6% and mamalian tubercle bacilli in 4.41 of the 248 tuberculous swine lesions examined.i Mixed infections involving both avian and mammalian tubercle bacilli were found in 5.6% of the specimens. The swine from which these lesions were obtained were from Nebraska. In addition to these, they examined 14 lesions from Michigan swine and found M. gyi____uln in 92.97. and mixed infections in 7.1%. Similarly, high incidences of M. M infection were found in lesions of pigs from Illinois (Graham and Tunnicliff, 1926), Minnesota (Feldman, 1938a; Feldman, 1939; Feldman and Karlson, 1940) and the North Central states (Crawford, 1938). Mohler and Washburn (1908) produced tuberculosis in swine experi- mentally by feeding them viscera of tuberculous chickens. Schalk g; 31. (1935) successfully transmitted tuberculosis to swine by contaminating their pens with droppings of tuberculous chickens and by feeding pigs tuberculous viscera from.chickens. They also*were able to infect swine by maintaining them on ground on which tuberculous chickens had been kept 2 years previously. The change of opinion regarding the main cause of porcine tubercu- losis is well illustrated by quotations from a U. S. Department of Agri- culture bulletin "Tuberculosis of Hogs" written in 1917 by Mohler and Washburn, and from a revision of the same bulletin (Mahler, 1938). From the 1917 circular we read: "Tuberculous cattle are the main source of tuberculosis in swine"; in 1938 it was revised as follows: "Tuberculous fowls are the main source of tuberculosis in hogs in the United States." 8 For many years, porcine tuberculosis was thought to be caused only by M. tuberculosis, M. REV—1.3. and M. M (Francis, 1958 ; Merchant and Packer, 1961; Jubb and Kennedy, 1963; Karlson, 1964). Karlson and Feldman (1940) had reported isolating rapidly growing, nonchromogenic, acid-fast bacilli with characteristics dissimilar to those of the aforementioned species. Because of their lack of virulence for laboratory animals and calves and because of the lack of lesions in the animals from which they were isolated, little significance was attached to these reports. However, Mallmann, Mallmann and Ray (1962) reported isolating non- chromogenic acid-fast bacilli from tissues of approximately 100 tuberculin- positive swine from a single herd. In most cases, these tissues contained gross tuberculoid lesions. These strains were classified as Group-III mycobacteria according to the classification scheme of Runyon (1959) by the methods of Mallmann, Mallmann and Robinson (1964). In an effort to determine whether atypical mycobacteria were responsible for tuberculosis-like lesions in swine from other geographic areas, such lesions were obtained from pigs originating from Michigan, Illinois, Missouri, Ohio, and Indiana. These tissues were examined bacteriologically. Eighty-five per cent of the acid-fast organisms isolated were M. m and 15% were Group-III mycobacteria (Mallmann _e_t_ 5L, 1962). Seaman, Franan and Will (1963) also isolated nonchromogenic acid- fast bacilli from tuberculous swine. They compared 43 stains isolated to 10 strains of the Battey bacillus (Group-III, human origin) and to 10 strains of M. M. They concluded that there was a close relationship among swine and himsn Group-III organisms and M. avium. 9 Karlson (1964) pointed out that in 11 reports of bacteriologic studies conducted on tuberculous lymph nodes of swine, the authors had not been able to identify a causative agent in from 11 to 61.5% of the samples examined. He says that this may be due to: l) inadequate technique, 2) the healing of the lesion, or 3) that the lesion may not be due to infection with tubercle bacilli. He drew attention to the reports of European and American workers who isolated Cogzgebacterimmleggi,repeatedly from such lesions, sometimes alone and sometimes concurrently with M. ggigg. He says, "... localized lesions (of 9, egg; infection) ... cannot be easily differentiated from tuberculous processes either macroscopically or histologically." Upon reviewing the works cited by Karlson (1964), i.e., the works of van Es and Martin (1925), Graham and Tunnicliff (1926), Mitchell, Walker and Humphreys (1934), MbCarter, Beach and Hastings (1935), Crawford (1938), Feldman (1938a, 1939), Feldman and Karlson (1940), Pullin (1946) and Bunkier (1946), one wonders whether the methods used would have demonstrated atypical mycobacteria, if present. All of the workers relied heavily upon animal inoculation for primary isolation. Even where artificial media were used, atypical colonies may not have been thought significant. In commenting about his failure to isolate tubercle bacilli from.4 specimens, Feldman (1938a) says: "Is it possible that unidentified acid-fast bacilli exist which are responsible for a tuberculous disease in swine? The failure of bacteria to grow on a culture mediumiwhich is suitable for ordinary forms of tubercle bacilli and the failure of emulsions made from morbid tissue to induce lesions in rabbits or guinea pigs can hardly be attributed to the infective agents' being 10 nonviable or avirulent. The lesions in the swine were of a generalized and progressive nature sug- gestive of marked virulence or exceptional suscepti- bility on the part of the respective swine." The isolation of atypical mycobacteria is not new. Shortly after Koch (1882) discovered the tubercle bacillus to be the causative agent of tuberculosis, Alvarez and Tavel (1885), Nocard and Roux (1888), and Ferran (1897) reported isolating acid-fast organisms with characteristics atypical of those of Koch's bacillus. Similar reports are repeatedly found (Bram, 1909; Frey and Hagan, 1931; Pinner, 1935; Baldwin, 1942; Buhler and Pollak, 1953; Runyon, 1959; Atwell and Pratt, 1960; McCusker and Green, 1962; Mallmann, Mallmann, and Ray, 1962; Corpe, Runyon, and lester, 1963; Mallmann, Mallmann and Robinson, 1964). Some of these organisms can be classified as one of the species of mycobacteria as set forth in the 7th edition of Bergey's Manual of Determinative Bacteriology (Breed, Murray, and Smith,'1957). Others cannot be so classified and have been called atypical (Pinner, 1935), anonymous (RunyOn, 1959), or unclassified (Corpe £11., 1963). Classification of mycobacteria is difficult because of their variable characteristics. Xalaburder (1961), writing on the subject of classifi- cation of mycobacteria, said: "The only thing about them.(mycobacteria) that is abso- lutely typical is their great plasticity and adapta- bility to an extreme variety of environments. ... Bacteriologists have established their systems of classification on the basis of assigned, fixed, pre- cise and immutable characteristics to each group of organisms. This was necessary for didactic purposes in the purely descriptive stage of bacteriologic re- search. However, as the agreement was unilateral, the 11 result that the bacteria, not aware of this con- venient human arrangement, continued their normal biologic processes, which consisted of a steady evo- lution and adaptation to the greatest variety of environmental conditions, so that now there is great discrepancy between the real state of affairs and that envisaged by our well-intentioned blueprints." The same can be said about any biologic classification. Classifi- cation is essential for comunication about and study of biology, but one mat remember that evolutionary and adaptive changes are constantly taking place. Acid-fast organisms, both classified and unclassified types, are almost ubiquitous. Brem (1909) reported their frequent occurrence in samples fran many inanimate sources. Frey and Hagan (1931) were success- ful in isolating acid-fast organisms from soil samples from many areas of the United States even though their techniques precluded the growth of many mycobacterial species. Pinner (1935) reported isolating acid-fast organisms from water taps and tap water. He wrote, "Acid-fast organisms have been isolated from almost any material properly scrutinized." This was confined by the work of Karlson (1958). Traum (1916) reported observing acid-fast organisms in the lesions of "skin lesion tuberculosis" of cattle and later (Trams, 1919) isolated atypical acid-fast organisms from such lesions. Frey and Hagan (1931) hypothesized that acid-fast bacteria from the soil might be the cause of these "skin lesions" as well as being the sensitizing agent causing the no-gross-lesion tuberculin reactions. Daines and Austin (1932) also iso- lated atypical acid-fast organisms from "skin lesions". Pinner (1935) isolated atypical mycobacteria in about 1% of the cultural work then being done at the Desert Sanitarium and Institute of Research in Tucson, Arizona. 12 In some, but not all, cases , M. tuberculosis was also isolated from these patients. Baldwin (1942) reported the clinical, bacteriologic, and tuberculin-sensitivity findings of a case of "pulmonary tuberculosis-like disease" associated with a "nonpathogenic acid-fast bacillus". Buhler and Pollak (1953) also reported 2 cases in human beings due to infection with atypical acid-fast organisms. Following this, reports of infections in human beings with atypical mycobacteria increased in frequency (Timpe and Runyon, 1954; Wood 9; $1., 1956; Crow .e_t; $1., 1957): Little or no significance was attached to the role of atypical acid- fast organisms in disease of man and other animals until the late 1950s and early 1960s. This was probably mainly due to 3 factors: 1) the high incidence of disease due to M. tuberculosis, M. m, or M. 2.21.2! which masked the relatively low occurrence of atypical mycobacterial infections; 2) atypical mycobacteria either are not pathogenic or vary in their pathogenicity for laboratory animals, such as guinea pigs, rabbits and chickens (Pinner, 1935; Baldwin, 1942; Buhler and Pollak, 1953; Pollak and Buhler, 1955; Wolinsky gt; 9_l.., 1957; Runyon, 1959; Mallmann, Mallmann and Robinson, 1964). These animals were used exten- sively for classification and determining the pathogenicity of myco- bacteria (Breed e_t_:_ $1., 1957; Runyon, 1959); 3) mycobacteria are so widespread in nature that only the classical pathogenic species were considered capable of causing disease. Karlson (1958) pointed out the difficulties in the classification of atypical mycobacteria with classification schemes existing at that time. Runyon (1959) studied atypical mycobacteria which were associated with 13 disease in human beings and preposed a system of classification. Based on colonial morphology, pigmentation and growth rate, he divided the atypi- cal mycobacteria into 4 groups: viz., Group I (photochromogens), Group II (scotochromogens), Group III (nonphotochromogens) and Group IV (rapid growers). Although Runyon's system of classification has been widely accepted and used, it has the major shortcoming of not separating pathogenic strains from nonpathogenic strains as was pointed out by Runyon (1959). He says that the guinea pig can no longer be used alone for determining the pathogenicity of strains for man and that other species must be found for the purpose. Based on the work of Lester (1939) and Kite, Patnode and Reed (1952), Kubica 2§_el, (1960) devised a method for virulence determination. With their method, 0.1 mg., 0.01 mg. or 0.001 mg. of the culture was injected intracutaneously in the abdominal region of guinea pigs., Cultures pro- ducing ulcers at the inoculation sites were considered to be pathogenic. Pollak and Buhler (1955), Durr 5;. l. (1959), and Feldman (1963) were able to produce disease in.hamsters with atypica1.mycobacteria suggesting the possible use of this animal either alone or in combination'with other species to determine pathogenicity. The problem of trying to associate pathogenicity in primary hosts with that in laboratory animals is difficult. Because of isolations of acid-fast organisms from apparently healthy human beings (Edwards and Palmer, 1959; Atwell and Pratt, 1960), and from normal animals (Malhmann, 1963), and because of the existence of apparently nonpathogenic atypical 14 mycobacteria (Mallmann g£,§;,, 1963; MCGavin, 1964), one can never be certain that the organism isolated was actually the cause of a lesion. Also, as pointed out by Mallmann, Mallmann and Robinson (1964), repeated transfer on media or inoculation into animals can change the virulence of a mycobacterial strain. A statement by Pinner (1935) regarding this matter is still applicable: Just "Classification as to pathogenicity is hampered by the fact that the term 'pathogenic' is nearly meaning- less unless strictly defined in terms of animal species, dosage, time interval between infection and pathological examination; and, most important of all, there is no general agreement on‘what consti- tutes disease in infected animals. If any demon- strable tissue alterations be called disease, and accordingly any organism that causes them is considered pathogenic, then there are no apathogenic acid-fast organisms. To stipulate a minimal dosage that must produce disease, in order to admit the organism into the classification 'pathogenic' is totally arbitrary. To assign the term pathogenic only to those micro- organisms that cause progressive disease, and to exclude all those that produce self-healing lesions, ‘would exclude a major portion of all so-called patho- genic (non-acid-fast) organisms. But such prOpOsals are on record. A fairly clear-cut distinction can be made by establishing whether a given microorganism causes lesions, progressively or retrogressive, in serial transfers from animal to animal. If this cri- terion is used to differentiate between pathogenic and saprophytic acid-fast bacilli it is apparent that all nonwmammalian acid-feats belong in the Baprophytic group, although it has probably never been settled whether or not some acid-fast isolated from cold- blooded animals are, in the sense specified, patho~ genic for the respective species." as applicable is the statement of Kubica g; a}. (1960): "The fact that it has been impossible consistently and repeatedly to fulfill Koch's postulates as regards the anonymous and isoniazid-resistant acid-fast bacilli is no reason why we should negate their flmportance in human disease." 15 The lesions produced by atypical mycobacteria appear to be impossible to differentiate grossly or histologically from those caused by g. tubercu- lo_s_i_s_. Corps and Stergus (1963) conducted a study wherein 27 pathologists who were interested in tuberculosis studied duplicate sets of 25 sections from human surgical specimens. Either 1!. tuberculosis or Group-III myco- bacteria had been isolated from each of these specimens as well as from the sputum of the patient. (h each slide they could choose one of the following classifications: l) the histologic picture was compatible with tuberculosis due to g. tuberculosis; 2) the histologic picture was compatible with tuberculosis due to the Battey strain, Group III, nonphotochromogens; 3) the histologic picture was compatible with tuberculosis but that it was impossible to determine the strain; 4) nontuberculous. The pathologists indicated that they could not tell which strain was the causative agent in 532. of the choices. Twenty-nine per cent of the choices indicated that the lesions were due to AI. tuberculosis infection. Only 38% of these were correct. Nontuberculous disease was indicated in 62 of the instances, all of which were in error. The results confirmed the authors' previous impression it is impossible to accurately differen- tiate histologically between lesions caused by Li. tuberculosis and those caused by Group-III mycobacteria. Corpe, Runyon and Tester (1963) reported their inability to differen- tiate, histologically, lesions caused by infections with any of the 4 groups of atypical mycobacteria. These lesions were similar to those caused by .13. tuberculosis. Crow 93; 51... (1957) reported similar findings. l6 McCavin (1964) reported that, while atypical mycobacteria varied in their ability to produce lesions when injected intradermally into calves, when lesions were produced it was impossible to differentiate them histo- logically from those caused by g. bovis infections. Feldman (1960a) noted difficulty in differentiating the lesions of tuberculosis from other infectious granulomas. He pointed out the impor- tance of suitable bacteriologic studies to determine the cause of a lesion. He stated (Feldman, 1938): "The hog occupies an unfavorable position biologi- cally as it is susceptible to all three forms of the tubercle bacillus, and provided that the Opportunity for exposure exists, one can safely assume that the frequency of tuberculosis in hogs and the type of tubercle bacillus responsible for the lesions con- stitute a rather accurate index of the amount of tuberculosis in fowl, manuals, and human beings in any particular locality." Consonant with this statement, one cannot help but wonder about the significance of the finding of atypical mycobacteria in the tuberculous lesions of swine and of similar findings in man and cattle. It is possible that the infections come from a comon source yet unidentified or that interspecies transmission may be occurring. MATER IAIS AND METHODS Egeriment a1 Animals Pigs were obtained at various times from 1 tuberculin-negative herd. They were Yorkshire-Berkshire-Landrace crossbred animals approxi- mtely 6 to 8 weeks of age when purchased. They were fed a standard growing ration obtained from the Department of Animal Husbandry, Michigan State University, and water a; libitum. The animals were housed in heated isolation rooms, several animals per room (Table 1). They were maintained without bedding on concrete floors, their refuse being washed into floor drains daily. All persons entering the rooms wore rubber suits and boots which were washed in disinfectant" after each use and left in the anteroom imediately adjacent to each animal room. All personnel wore disposable gloves, caps and masks which were used only once and in only 1 room. Cultures The cultures were from the collection of the Tuberculosis Research Project, Michigan State University, and were as follows: 1. Mycobacterium m, swine origin: 81-0. 2. Group-III mycobacteria, swine origin: 167C1-l, 17201-1, 186C-l, and 19302-1. 3. Group-III mycobacteria, pen origin: 15D, 192w. *Torsite, The Dow Chemical Company, Midland, Michigan 17 l8 4. Mcobacterium gi___um, swine origin: 206-2 5. Mycobacterium m, laboratory strain The atypical mycobacteria were classified according to the system of Mallmann, Mallmann and Robinson (1964). The Group-III cultures of swine origin had produced ulcers at the site of intradermal inoculation of guinea pigs while those of pen origin did not. The M. bovis, M. avium (swine origin), and the Group-III mycobacteria (swine origin) had been isolated from tissues of tuberculous swine. The Group-III mycobacteria of pen origin were from pens where swine infected with Group-III myco- bacteria had been housed, isolated a week after the pens had been cleaned and disinfected* using a high-pressure apparatus. The l_4. 935L112 (laboratory strain) had been maintained for many years on artificial media as a stock culture. It was, however, still virulent for chickens. The culture numbers are those of the Tuberculosis Research Project and were used here so that easy reference can be made totheir work. The derivation of l of these numbers (167C1-1) will be explained so that they can be better understood. Culture 16701-1 was isolated from tissues of the 167th animal examined by the project in 1961 (case number 167-1). The "CI" indicates that it was the 1st of at least 2 colony types (which appeared to be different on primary isolation) from the pool of lymph nodes fran the peritoneal cavity (designed as "C pool" by the Project personnel). The letters "D" and "W" used in the identification of the Group-III mycobacteria of pen origin refer to whether the swab used to collect the sample was wet or dry. *Torsite, The Dow Chemical Company, Midland, Michigan 19 Administration of mcobacteria The pigs were maintained in the isolation rooms 2 to 3 weeks before the administration of mycobacteria. Each inoculated pig was given 2 mg. wet weight (approximately 2 x 108) cells suspended in 0.2 ml. Dubos broth base without Tween 80* but with 0.52. dextrose added. Intra- dermal inoculations were made on the lateral surface of the left foreleg, just distal to the carpus, using a 3/8-inch, 26-gauge hypodermic needle and syringe. The oral administrations were made after withholding feed from the animals for 1 day. Each pig was fed from a sterile pan a cohesive mixture of the ration and sterile molasses; 2 mg. wet weight of mycobacteria were incorporated into this mixture. Each animal was seen to eat all of the mixture. Uninoculated pigs were housed with pigs to which mycobacteria were administered to determine if the organisms were transmitted by contact. Uninoculated negative control animals which served as controls were maintained like the inoculated pigs in adjacent isolation rooms. In some instances, if these pigs had shown no signs of disease and were tuberculin negative, they were sold for slaughter in order to salvage their meat value. In this event they were inspected by careful and detailed examination for gross lesions. Clinical Examinations The pigs were observed daily for signs of disease. The sites of intradermal inoculation were carefully examined and blood samples were *Difco Laboratories, Inc., Detroit, Michigan 20 drawn.periodically from the anterior vena cava during the course of the experiment. Serum obtained from these samples were used for serologic studies being conducted concurrently. Patholgic Technigues The animals were anesthetized with intravenous injections of pento- barbital sodium" and transported to a necrOpsy room. There they were killed by exsanguination. Blood samples were collected for hematologic and serologic studies. The necropsy technique was based on that of Jones and Gleiser (1954) as modified by McGavin (1964). Sections of the liver, heart, lung, both kidneys, spleen, and ileum were put in 107. buffered, neutral formalin and Zenker's fluid (without acetic acid) while the necropsy was being performed. The following lymph nodes and the skin inoculation site, where appli- cable, were dissected free, identified and put into plastic bags for transportation to the laboratory: right and left submaxillary, right and left parotid, right and left lateral retropharyngeal (atlantal), right and left medial retropharyngeal (parapharyngeal), anterior mediastinal, posterior mediastinal, right and left bronchial, hepatic, gastric, mesen- teric, right and left prescapular, right and left prefem‘oral, right and left popliteal, internal iliac, right and left deep inguinal, right and left external inguinal and any others found affected. These tissues were cut in the laboratory under a bacteriologic hood and observed for gross lesims. Sections, 3 to 4 m. thick, were fixed in 10% buffered, neutral - formalin. *Halatal Solution, Jensen-Salsbery Laboratories, Kansas City, Mo. 21 The tissues were infiltrated and embedded in a tissue embedding medium" by techniques suggested in the U. S. Armd Forces Institute of Pathology Manual of Histologic and Special Staining Technics (1957), with chloroform being used as the clearing agent. Sections were cut at 6 microns' thickness and stained with the new fuchsia-hematoxylin-eosin method of Willigan, Garric and Trosko (1961). The tissues were evaluated on the basis of the presence and charac- teristics of the gross and microscOpic lesions and the location and number of les ions found. Bacteriolgic Technigues Tissues to be examined bacteriologically were carefully handled so as to minimize contamination. They were transported in plastic bags to the laboratory, where the attached adipose tissue was trimmed from them. They were washed 5 times for 5 minutes each time in 500 p.p.m. sodium hypochlorite solution. The tissues were sectioned aseptically and samples taken for histOpathologic and bacteriologic examinations. The lymph nodes were pooled for bacteriologic examination as follows: A. Lymph nodes of the head and neck region B. Anterior and posterior mediastinal and bronchial lymph nodes C.' Hepatic, gastric, mesenteric and colic lymph nodes D. Left prescapular lymph node E. Lung F. Skin inoculation site 1. Other tissues (cultured separately and identified) *Paraplast, Arthur H. Thomas Co., Philadelphia, Pennsylvania 22 Following sectioning, the tissues for bacteriologic examination were ground in nutrient broth? with a Waring Blendor or mortar and pestle. Ten ‘milliliters of the resulting homogenate were mixed with 10 ml. 41 NaOH, shaken and allowed to stand for approximately 15 minutes. The mixture was neutralized with 2% H01, centrifuged and the supernatant fluid decanted. The sediment was used to seed 6 tubes each of Lowenstein-Jensen Medium,* * HuddlebrOOk 7H10 agar,* and Dubos Oleic agar. The media were incubated at 35 C and observed biweekly for growth for 3 months. Tuberculin Test Technigues The animals were tuberculin-tested approximately 1 week before necropsy and, in certain cases, during the course of the experiment. The tests were conducted as follows: the thickness of the ear was measured by means of a vernier caliper** and the thickness recorded. Then, 0.1 ml. avian tuberculin*** was injected intradermally on the right ear and 0.1 m1. ummumlian tuberculin# was injected on the left ear. The injection sites were examined approximately 48 hours later. The thickness of any detectable response was measured and recorded. *Difco Laboratories, Inc., Detroit, Mflchigan **Scientific Products, Evanston, Illinois ***Tuberculin, avian, intradermic, produced for the Agricultural Research Service (ARS), USDA #Tuberculin, mammalian, intradermic, produced forthe ARS, USDA 23 Hemat olgic Technigue A blood sample was collected during exsanguination into a tube con- taining dipotassium ethylenediamine tetraacetate (EDTA). This was used for hemoglobin, hematocrit and total leukocyte determinations. 24 TABLE 1. Index of pig number, housing schedule, route of administration, culture number and source, culture classification, duration of experiment, and page number in results. Pi: GroupT Culture No. Culture ' Duratib:2 Page No. No. Route and Origin Classification in Days No. 2-2 1 m3 81-0;Swine a. bovis 57 29 2-1 1 ID 81-0;Swine M. bovis 66 32 2-4 1 ID 81-0;Swine M. 2.92.1.9. 112 35 3-4 2 ID 167C1-1;Swine Group 1114 59 37 1-3 2 ID 167C1-l;Swine Group III 107 42 3-2 2 ID l86C-l;Swine Group III 66 43 3-1 2 ID l86C-l;Swine Group III 107 44 3-3 2 Contacts 59 44 1-4 3 ID 172C1-l;Swine Group III 65 45 1-1 3 ID 17201-1;Swine Group III 113 46 2-3 3 ID 19302-1;Swine Group III 65 47 1-6 3 ID 193C2-l;Swine Group III 113 48 1-5 3 Contact 113 49 5-3 4 ID Lab. strain 14. 93$! 82 49 5-4 4 Contact 82 50 5-2 4 ID Lab. strain M. M 138 51 5-5 4 Contact 138 52 1All pigs of each group housed in a single and separate isolation room. 2Days after administration of the organism when necropsied. 3Intradermal inoculation. (Runyon Group-III mycobacteria. 5Contact pen-mate which was neither inoculated with nor fed the culture. TABLE 1--cont inued 25 Pig Group Culture No. Culture Duration Page No. No. Route and Origin Classification in Days No. 591 4 :1) Lab. strain g. m 215 53 5-6 4 Contact 215 54 10-3 5 ID 206-2;Swine M. avium 43 54 10-1 5 ID 206-2;Swine M. avium 57 56 10~4 5 ID 206-2;Swine M. avium 113 59 10-2 5 ID 206-2;Swine M. avium 113 59 10-5 5 ID 206-2;Swine M. svium 175 60 10-6 5 ID 206-2;Swine M. avium 175 61 10-7 5 Contact 182 61 10-8 5 Contact 182 62 161-3 6 ID 15D;Pen Group III 138 62 161-4 . 6 ID 19 W;Pen Group III 138 63 161-6 6 ID 15D;Pen Group III NGL5 62 161-2 6 ID 192W;Pen Group III NGL 63 161-7 6 Contact NGL 63 161-8 6 Contact NGL 63 161-9 6 Contact NGL 64 7-1 7 Oral 81-0;Swine M. ME! 79 64 7-6 7 Contact 79 66 7-2 7 Oral 81-O;Swine M. by; 134 67 7-5 7 Contact 134 68 5N0 gross lesions when examined at slaughter. TABLE l--cont inued 26 Pig Group Culture No. Culture Duration Page No. No. Route and Origin Classification in Days No. 7-3 7 Oral 8l-O;Swine M. bay}; 203 69 7-4 7 Contact 203 70 6-1 8 Oral 17201-1;Swine Group III 78 71 6-6 8 Contact 78 72 6-2 8 Oral 172C1-l;Swine Group III 136 72 6-5 8 Contact 136 73 6-3 8 Oral 17201-1;Swine Group III 206 73 6-4 8 Contact 206 74 8-2 9 Oral Lab. strain M. 2111!.“ 52 76 8-1 9 Contact 52 76 8-3 9 Oral Lab. strain M. a_vi___um 108 76 8-5 9 Contact 4108 76 8-4 9 Oral Lab. strain M. £1112 169 77 8-6 9 Contact 169 77 9-2 10 Oral 206-2;Swine M. m 57 77 9-1 10 Oral ' 206-2;Swine M. aviun 57 78 9-3 10 Oral 206-2;Swine M. aviun 115 78 9-4 10 Oral 206-2;Swine M. avium 115 79 9-5 10 Oral 206-2;Swine M. svium 171 79 9-6 10 Oral 206-2;Swine M. avium 171 80 9-7 10 Contact 177 81 9-8 10 Contact 177 80 1-2 11 Contro17 108 81 7Negative control to which no culture was administered and which was not in contact with exposed pigs. TABLE 1--cont inued 27 Pig Group Culture No. Culture Duration Page No. No. Route and Origin Classification in Days No. 2-6 11 Control 108 81 2-5 11 Control 108 81 4-1 12 Control NGL 81 4-2 12 Control NGL 81 11-1 13 Control NGL 81 11-2 13 Control NGL 81 11-3 13 Control NGL 81 11-4 13 Control NGL 81 11-5 13 Control NGL 81 11-6 13 Control NGL 81 11-7 13 Control NGL 81 11-8 13 Control NGL 81 11-9 13 Control NGL 81 11-10 13 Control NGL 81 RESULTS Seventy-five pigs were used in this study. Mycobacteria @. 29333, M. 911-1229 or Group III) were administered to 39 pigs either intradermally or orally, while 20*were uninoculated pen-mates of these animals. The remaining 16 pigs were non-contact uninoculated control animals. .Unless otherwise noted, all pigs were apparently healthy through- out the experiment and were in a good state of nutrition at the time of necrOpsy. Immediately following is a description of the findings from each pig organized according to culture and route of administration. The findings from the uninoculated penmmates are included with those of their pen-mates. Bacterial isolations and lesions are summarized in Table 2, which follows the detailed descriptions. In all cases the organisms isolated resembled culturally the organisms administered. The dimensions of the lesions found at the inoculation site upon clinical examination‘were obtained by measuring across the circular to oval lesions and, therefore, refer only to the area of the lesion. The 3rd dimensions are lacking since the height or thickness of the lesions could not be determined accurately during life because of their location over the carpus, their limited elevation, and the impossibility of deter- mining at that time how deeply the lesion extended into the underlying tissue. 28 29 Ag Intraderma 1 Inoculat ions Culture 81-0, M. bovis, swine origin Pig 2-2 Clinical obserzations. Eight days after inoculation the inoculation site was swollen (25 x 30 m.) and covered with an eschar. On the 15th day after inoculation the swelling at the site was 20 x 30 11:11., ulcerated and a purulent exudate was draining from it. Twanty-three days after inoculation the lesion was swollen and hard (25 x 40 um.) and still dis- charging a purulent exudate. By the 35th day the swollen area was 20 x 25 m., denuded and dry. Thirty-eight days after inoculation the lesion was 20 mm. in diameter and dry. On the 44th day there was a very slight swelling (20 x 25 m.) and a dry ulcer at the inoculation site. By 52 days the lesion consisted of a slightly swollen area (20 x 25 m.) with a central, dry eschar. Necrgpsy findiggg (57 days after inoculation). Generalized lesions of tuberculosis were found, with the following lymph nodes containing gross lesions: anterior cervical, left submaxillary, lateral retro- pharyngeal, middle cervical, anterior and posterior mediastinal, bronchial, mesenteric, hepatic, left prescapular and nodes of the lumbar chain. With the exception of the left submaxillary and anterior cervical lymph nodes, the lesions were multiple, white, 1- to 2-nln. foci scattered throughout the nodes. In the left submaxillary lymph node, multiple white caseous foci up to 5 m. in diameter were found. The center of the anterior cervical lymph node was filled with yellowish caseous material. 30 The lesions in the hepatic lymph nodes were calcified. Multiple white foci, l to 2 mm. in diameter, were scattered throughout the parenchyma of the liver. An ulcerated area 25 m. in diameter with a granulating base, was found in the skin at the site of inoculation. Histgatholgic findipgs. Microscopic granulomas were found in all lymph nodes which had gross lesions. These granulomas were similar, having central areas of caseation necrosis surrounded by macrOphages. Many of these foci had become confluent and at times the caseation necro- sis filled the center of the lymph node. In the left prescapular node, the granulomas were unencapsulated groups of macrOphages, some with cen- tral caseation necrosis (Figures 1 and 2). The left parotid lymph node, in which no gross lesions were found, contained a few scattered unencapsu- lated granulomas consisting predominantly of macrOphages and giant cells. The lumbar chain of lymph nodes contained numerous scattered focal lesions varying from accumulations of a few macrOphages to granulanas with cen- tral caseation necrosis surrounded by macrophages and small amounts of connective tissue. Numerous small granulomas made up of small groups of macrOphages were found scattered throughout the right prescapular lymph node. No necrosis or encapsulation was found. The supramsmary lymph nodes contained numerous scattered discrete granulomas consisting of small groups of macrophages and occasional giant cells. In addition, there were a few larger granulomas with caseous centers surrounded by macrophages and some connective tissue. The degree of encapsulation of the granu- lomas varied from slight to marked in the anterior cervical, middle cervi- cal, left submaxillary, right and left medial retropharyngeal, left '- M“: -..L.'_~ 'J?'" '13“ .95 ’ v- -+‘. u .- 2‘ 7 '1 .~ \ . 8'5? Figure 1. An unencapsulated, noncaseous granuloma found in the left prescapular lymph node of Pig 2-2, inoculated intradermally with Culture 81-0, M. bovis, swine origin. New Fuchsin - H & E. x75. Figure 2. Left prescapular lymph node of Fig 2-2, inoculated intradermally with Culture 81-0, M. bovis, swine origin. Note area of coagulation necrosis and necrotic cells (1), surrounded by macrophages (2), and the absence of encapsulation. New Fuchsin - H & E. x187. 32 parotid, right bronchial, and hepatic lymph nodes. The skin ulcer from the site of inoculation contained much connective tissue, among which were granulomas composed of macrophages and having, in some instances, centers of caseation necrosis. Multiple scattered discrete or confluent granulanatous foci were found in the lungs. Some of these areas were caseous or caseocalcareous. Early attempts at encapsulation were present. Granulanas with caseocalcareous centers were fomd in the liver. Some areas of necrosis were surrounded by macrophages and lymphocytes. These lesions were slightly encapsulated. In the spleen, a few small granu- lomas with caseous centers were found. Early encapsulation attempts were in evidence. Pig 2-1 Clinical observations. Eight days after inoculation the inoculation site was reddened (35 x 40 m.) and ulcerated with a purulent material exuding from it. By 15 days after inoculation, there was a 40 x SO-nm. swelling with s purulent ulcer. There was a 40 x 55-11-11. swelling with s 20 x 20m. suppurating ulcer present 23 days after inoculation. When examined 35 days after inoculation, there was a 50 x 55m. swelling with a dry ulcer found at the inoculation site. Three days later, the size of the lesion was 45 x 50 m. Forty-four days after inoculation, the skin lesion was a 30 x 40-11111. swelling with a central dry ulcer. Fifty- two days after inoculation, a 50-m.-diameter swelling with a central eschar was found . 33 Necrogsy findiggs (66 days after inoculation). One- to 2-mm. white foci were found in the following lymph nodes: left parotid, left sub- maxillary, one node of the lumbar chain, anterior and posterior mediastinal, right bronchial and hepatic lymph nodes. Multiple white foci varying in size from 1 m. in diameter to l- x 4-m. areas were found in the left prescapular lymph node. The liver arr! spleen contained white foci, 2 to 3 m. in diameter, throughout the parenchyma. The skin lesion consisted of a swollen area 20 m. in diameter with a central, lO-nln.-diameter ulcer. On cross section, the ulcer appeared shallow. Histgpatholggic findings. Discrete and confluent granulomas were found in the parotid, retrOpharyngeal, left submaxillary, anterior and posterior mediastinal, right bronchial, lunbar, hepatic, mesenteric, and prescapular lynph nodes. Caseocalcareous lesions were found in all lymph nodes except the right prescapular. The degree of encapsulation varied somewhat, but unencapsulated lesions were found in all of these tissues. The cellular components were macrophages and occasionally giant cells. Hyperkeratosis, acanthosis, and a small ulcer were found in the skin at the site of inoculation. Numerous well-encapsulated granulanas were found in the dermis. Caseation necrosis and calcification were found in the center of l of these granulomas. Groups of lymphocytes and neutro- phils were found in the lesion. Unencapsulated granulomas with caseocal- careous centers surrounded by macrOphages and lymphocytes were found in the liver. Well-encapsulated granulomas with caseocalcareous centers were found in the spleen (Figure 3). In the lung, numerous scattered 34 Eigure 3. Capsule of a well-encapsulated granuloma found in the spleen of Pig 2-1, which was inoculated intra- dermally with Culture 81-0, M. bovis, swine origin. New Fuchsin - H & E. x187. 35 granulomatous foci, some with caseocalcareous centers, were found (Figures 4 and 5). These contained macrophages, eosinOphils and lymphocytes. Little encapsulation was found. Pig 2-& Clinical observations. Eight days after inoculation, the inoculation site contained a circular lesion 25 mm. in diameter which was reddened with a necrotic center. By 15 days after inoculation the lesion consisted of a 25 x 35-mm. swelling with purulent material draining fran a central ulcer. Twenty-three days after inoculation the skin lesion was a 25 x 40-mm. swelling with a 15 x 20m. draining central ulcer. The lesion was 30 x 45 mm. in size and reddened 35 days after inoculation. At 38 days after inoculation the lesion was 30 x 35 m. in size. At 44 days, the lesion was a 30 x 40-nsn. swelling with a dry, central ulcer. When examined at 52 days, the lesion was slightly swollen, 30 x 30 am. in size, dry and covered with an eschar. Following this the lesion healed. Necrmy findings (112 days after inoculation). Scattered l- to 3-mm., yellowish foci were found in the supramamary, right submaxillary, right parotid, and medial and lateral retropharyngeal lymph nodes. The left submaxillary lymph node was enlarged and filled with a reddish-yellow caseous mass. The gastric, anterior and posterior mediastinal, and the bronchial lymph nodes were enlarged and contained both discrete and con- fluent caseocalcareous foci. The left prescapular lymph node was 40 x 30 x 20 m. and filled with discrete and confluent caseocalcareous foci. Almost all mesenteric nodes contained caseocalcareous lesions 1 mm. in Figure 4. A granuloma with a caseocalcareous center (1) found in the lung of Pig 2-1, which was inoculated intradermally with Culture‘8l-0, M. bovis, swine origin. New Fuchsin - H & E. x75. Figure 5. Noncaseating, unencapsulated granulana found in the lung of Pig 2-1, which was inoculated intra- dermally with Culture 81-0, M. bovis, swine origin. New Fuchsin - H & E. x187. 37 diameter or larger. The hepatic lymph nodes were enlarged and filled with discrete and confluent caseocalcareous foci. The skin lesion was 60 x 30 m. in area and 15 m. thick and contained yellowish caseous foci distributed throughout. The lung, liver, and spleen contained many yellowish caseous foci, l to 6 nan. in diameter. Histggtholggic findiggs. Numerous granulomas were found in the following lymph nodes: submaxillary, medial and lateral retropharyngeal, anterior and posterior mediastinal, bronchial, hepatic, gastric, mesen- teric, supramamary, left prescapular and right parotid. These lesions ranged from small, unencapsulated groups of macrOphages to large, well- encapsulated granulomas with caseocalcareous centers. The caseous material almost filled the left submsxillary, supremamnary, and left prescapular lymph nodes (Figure 6). Well-encapsulated granulomas with necrotic centers were found in the right prefemoral and left pOpliteal lymph nodes. Numerous scattered granulomas were found in the lungs, liver (Figure 7) and spleen. These varied from small groups of macro- phages, lymphocytes, and a few giant cells to large, well-encapsulated granulomas with caseocalcareous centers. The dermis was thickened and contained numerous scattered granulomas, many with caseous or caseocal- careous centers. Cellular response included primarily macrophages and lymphocytes . Culture 167C1-1, Group III, swine origin Pig 3-4 Clinical observations. On the 8th day after inoculation there was a hard circumscribed lesion 20 m. in diameter with a central eschar. #rr‘ Figure 6. Well-encapsulated granulanas found in the left prescapular lymph node of Fig 2-4, which was inoculated intradermally with Culture 81-0, 5. bovis, swine origin. Note necrotic center (1) surrounded by macrophages and lympho- cytes (2) and dense connective tissue capsule (3). New Fuchsin - H & E. 1:75. Figure 7. Granulana with "daughter" tubercle found in the liver of Pig 2-4, which was inoculated intradermally with Culture 81-0, DJ. bovis, swine origin. Note caseocalcareous center (1), connective tissue capsule (2), and unencapsulated "daughter" tubercle consisting of macrophages and lymphocytes 39 Fifteen days after inoculation a 20 x 30-m. swelling was found which had a central ulcer with purulent exudate exuding from it. By 23 days after inoculation the lesion was 30 x 40 mm. in size with caseous material in the ulcerated area. On the 35th day the lesion was 30 nan. in diameter and moist. The lesion was a soft swelling, dry and scabbed, and was 35 x 40 mm. by the 44th day. Fifty-two days after inoculation a 25 x 40-min. swelling was present which had a small central eschar. Necrgsz finding (59 days after inoculation). Multiple white foci, l to 1.5 nm. in diameter, were found scattered throughout the left pre- scapular lymph node. They were more numerous in the middle portion of the node and under the subcapsular sinus. There was a raised lesion in the dermis which was yellowish and 10 Inn. in diameter. In the underlying subcutis a localized reddened area was found. Histgpatholggic findings. Several scattered small granulomas con- sisting of small groups of Langhans' giant cells and a few macrophages were found in the right submaxillary lymph node. In the left prescapular lymph node numerous granulomas were found which in some areas were becom- ing confluent. In many of the larger granulomas caseation necrosis and some calcification were found in the center (Figure 8). The necrotic areas were surrounded by macrophages, lymphocytes, some neutrophils, and eosinOphils. Unencapsulated small granulomas and daughter tubercles were also found (Figure 9). Multiple scattered focal granulanas consist- ing of groups of macrophages and giant cells were found in the bronchial lymph nodes and posterior mediastinal lynph nodes (Figure 10). Focal granulomas consisting of groups of macrOphages were found scattered Figure 8. Contiguous granulanas found in the left pre- scapular lymph node of Fig 3-4, inoculated intradermally with Culture 16701-1, Group-III mycobacteria, swine origin. Note necrotic areas (1) and dense connective tissue encapsu- lation (2). New Fuchsin - H & E. 1:75. Figure 9. Unencapsulated, noncaseous granulanss con- taining macrophages, giant cells and lymphocytes found in the left prescapular lymph node of Fig 3-4. New Fuchsin - H & E. x187. 41 Figure 10. Unencapsulated, noncaseous granulomas con- sisting of groups of macrophages and giant cells found in the right bronchial lymph node of Pig 3-4, which was inocu- lated intradermally with Culture 16701-1, Group-III myco- bacteria, swine origin. New Fuchsin - H & E. x187. 42 throughout the Spleen parenchyma. In the skin lesion, well-encapsulated granulomas maie up of macrophages and lymphocytes and sometimes central areas of caseation necrosis were found. Areas of lymphocytic infiltra- tion sometimes containing macrOphages and/or giant cells were found throughout the parenchyma of the liver. Pig l-Q Clinical observations. Eight days after inoculation the lesion at the site of inoculation was swollen, soft, 15 x 20 m. in size and had a central eschar. By 15 days the lesion was 20 mm. in diameter. A swelling 25 m. in diameter was found at the inoculation site on the 23rd day. Thirty-five days after inoculation the lesion was 20 m. in diame- ter, necrotic, and dry. At 44 days it was a soft 20 x 25-min. swelling. The lesion was 20 m. in diameter and covered with a dry eschar on the 52nd day, after which it healed. Necrgpsy findings (107 days after inoculation). One of the submaxil- lary lymph nodes was filled with a yellow caseous material. The skin was reddened and the dermis slightly thickened at the inoculation site. No other lesions were found. Histggatholgic findings. One of the submaxillary lymph nodes con- tained numerous scattered granulomas; sane were well-encapsulated and had caseocalcareous centers while others consisted of small groups of macro- phages and/or numerous giant cells. Occasional isolated groups of 2 or 3 giant cells accompanied by a few macrophages were found in the left pre- scapular lymph node. The only lesion found at the inoculation site was slight dermal thickening. 43 Culture 1866-1, Group III, swine origin Fig 3-2 Clinical observations. Eight days after inoculation a hard, 20 x 25-m. swelling was found at the inoculation site. On the 15th and 23rd days the swelling was edematous and 25 m. in diameter. 0n the 35th and 44th days after inoculation the skin lesion was edematous and measured 25 x 40 m. Fifty-two days after inoculation the lesion consisted of a swollen area 25 m. in diameter which was ulcerated with purulent material exuding from it . Necrgpsz finding (66 days after inoculation). The prescapular lyIph node contained numerous pale yellow foci l to 2 an. in diameter scattered uniformly throughout the tissue. In the skin an ulcer 2 m. in diameter was found. 0n section, this was reddened and appeared to be healing. Histogtholgic finding. Many scattered granulomas, consisting of macrophages and a few giant cells, were found in the left prescapular lymph node. Many of these granulanas had caseous or caseocalcareous centers. Sometimes the centers contained only a few degenerating neutro- phils. Encapsulation varied from slight to moderate. In the skin, hyper- keratosis, acanthosis, and dermal thickening were found. Discrete and confluent granulanas were found in the dermis. These granuloms con- tained macrOphages, lymphocytes, eosinophils, and neutrophils. Fig 3-1 Clinical observations. A hard and edematous swelling 15 x 20 m. in area was found at the inoculation site 8 days after inoculation. By 1.5 days after inoculation, the swelling was 20 x 50 m. in area. Eight days later the swelling was soft and measured 25 x 50 mm. Thirty-five days after inoculation the lesion was a 20 x 35m., soft swelling. Little change was noted at 44 days. By) 52 days the lesion had ulcerated and was 10 x 10 m. in area. Following this it healed. Necroggy findingg (107 days after inoculation). The left prescapu- lar lymph node was 20 x 25 x 16 m. and was filled with discrete and confluent, whitish, caseous areas 1 m. in diameter or larger. The skin at the inoculation site was possibly somewhat thickened. Histgpgtholggic findings. The left prescapular lymph node contained numerous lesions ranging from unencapsulated groups of macrophages to well-encapsulated granulomas with caseous or caseocalcareous centers surrounded by macrophages and lymphocytes. The larger, older lesions were becoming confluent. Some dermal thickening was noted in the skin at the site of inoculation. Pi - (Uninoculated pen-mate of animals inoculated with Culture 167C1-l or culture l86C-l) Clinical observations. No signs of disease were noted. Necrgpsz findings (59 days after inoculation of pen-mates). No gross lesions were found. 45 Histgpatholggic finding. No microscopic lesions were found. Culture 172C1-l, Group III, swine origin Pig 1:5 . Clinicgl observations. At 8 and again at 15 days after inoculation the lesion at the inoculation site was a 20 x 20m. swelling. Twenty- three days after inoculation the lesion was 25 x 30 m. and soft and had a central eschar. At 35 days after inoculation the lesion was a 20 x 30-m. swelling. By 44 days after inoculation the lesion was ulcerated, bleeding, and soft and 25 x 30 an. in size. Fifty-two days after inoculation there was a 25 x 30-min. swelling with a central eschar at the site of inoculation. Necrogz findings (65 days after inoculation). Numerous, scattered, l to 3-m. foci which appeared to be coalescing into lesions up to 10 um. or more in diameter were found in the left prescapular lymph node. The skin was thickened (30 m. in diameter) but did not appear to be ulcerated. (he caseous area about 3 m. in diameter was found in the thickened area. Histggatholgic findiggs. Numerous scattered granulanas were found throughout the hepatic lymph node. These ranged from small groups of macrophages to relatively large areas consisting of macrOphages and giant cells surrounding a central area of caseation necrosis. Several scattered granulomas consisting of small groups of macrophages with a few giant cells were found in the nodes of the lumbar chain. There were a few scattered granulomas consisting of small groups of macrOphages in 46 the posterior mediastinal lymph nodes. Numerous lesions were found in the left prescapular lymph node. These ranged from unencapsulated groups of macrophages and/or numerous giant cells to well-encapsulated granulomas with caseous or caseocalcareous centers surrounded by macro- phages and lymphocytes. The dermis was thickened by an increase in connective tissue. Fig 1-1 Clinical obserEtions. Eight days after inoculation the skin lesion was swollen and hard and 15 x 20 m. in area. At 15 days a 20 x 30-m. swelling with a central eschar was found. By 23 days after inoculation the lesion was 25 x 30 um. in area, ulcerated, and there was a purulent material exuding from it. Thirty-five days after inoculation there was a .25 x 25-min. swelling and serous exudation. By 38 days the lesion was 25 x 30 m. in area. Forty-four days after inoculation the lesion was 15 m. in diameter and had a dry, central eschar. By 52 days the lesion was healed . Necrogz findings (113 days after inoculation). Multiple discrete and confluent caseocalcareous foci were present in l of the submaxillary lymph nodes. There was a 30 1: 25-11111. area which was 12 m. thick in the dermis at the site of inoculation, but no caseous foci were found. Histgpatholggic findiggg. Numerous lesions ranging from small, slightly encapsulated groups of macrophages to large, well-encapsulated granulomas with caseous centers surrounded by macrophages and lymphocytes were found in a submaxillary lymph node. Many giant cells were found in 47 the left prescapular lymph node. Cells of the Langhans type were more numerous but foreign-body giant cells were also present. In sane places the giant cells were surrounded by macrophages. Acanthosis, some hyper- keratosis, a healing ulcer, and dermal thickening were found in the skin. There were a few scattered microscopic granulomas consisting of macro- phages, lymphocytes, and eosinophils surrounded by connective tissue. Culture 19302-1, Group III, swine origin Fig 2-3 Clinical observations. Eight days after inoculation there was a hard 30 x 40m. swelling at the inoculation site. By 15 days after inocu- lation the swelling was 30 x 40 m. in area. At 23 days it was 30 x 60 am. By 35 days the swelling was edematous, 30 x 50 nsn.. exuding serous fluid or weeping. Nine days later the swelling was 30 x 50 m. and soft. The lesion was an ulcerated, 30 x 45-mn. swelling and‘was exuding puru- lent material by 52 days. Necrgpsy findings (65 days after inoculation). The left prescapular lymph node contained numerous l- to 4-m., yellowish foci. A thickened area approximately 40 m. in diameter containing numerous yellowish caseous areas was found in the skin. This lesion contained an 11 x lS-m. ulcer. Histgatholggic findiggs. The left prescapular lymph node was almost filled with lesions ranging from unencapsulated groups of macro- phages and giant cells to large, well-encapsulated granulanas with caseous or caseocalcareous centers. Also a few isolated giant cells were found. 48 Hyperkeratos is , acanthos is , dermal thickening, few granulomas , and an ulcer were found in the skin at the inoculation site. Fig 1-6 Clinical observations. Eight days after inoculation a hard, 20 x 30-mm. swelling was present at the inoculation site. Seven days later the swelling was 25 x 35 am. At 23 days after inoculation there was a 25 x 35-mm. swollen area with a central eschar. By 35 days the lesion was ulcerated, dry, and measured 20 x 40 m. Three days later the swelling was 20 x 25 m. At 44 days, it still was 20 x 25 m. but was dry and had a central eschar. Fifty-two days after inoculation there was an eschar 10 um. in diameter at the site, but no swelling. After this the lesion healed. Necroggy findings (113 days after inoculation). A yellowish caseous lesion 5 x 5 x 8 m. and 3 lesions 2 am. in diameter were found in l medial retrOpharyngeal lymph node. One submaxillary lymph node contained many yellow, caseous, confluent or discrete lesions. The Opposite node was filled with yellowish, caseous material. A slight thickening of the dermis was found at the inoculation site, but no caseous foci were detected. Histgpathologic findings. Numerous scattered granulomas consisting of small groups of macrophages and/or giant cells were found in the medial retropharyngeal lymph nodes. These were sometimes associated with small accumulatims of necrotic neutrOphils. Numerous giant cells 49 were found scattered throughout the submaxillary lymph nodes. These were occasionally surrounded by macrophages. Numerous, scattered granu- lomas were found in the left prescapular lymph node. These ranged from small unencapsulated granulomas consisting of l or more giant cells or a group of macrophages and/or giant cells to well-encapsulated granulanas with caseocalcareous centers surrounded by macrophages and giant cells. Only hyperkeratosis and dermal thickening due to increased collagen were seen in the skin. Pig l-§ (Pen-mate of swine inoculated with Culture 172C1-l or 193C2-l) Clinical observations. No clinical abnormalities were seen. Necrogy findings (113 days after inoculation of pen-mates). No gross lesions were found. Histfltholggic findings. No microscopic lesions were found. 11. m, laboratory strain 11.8.5.2 Clinical observatigpg. Eight days after inoculation there was a slight swelling, 10 -. in diameter, at the inoculation site. At 14 days after inoculation there was a 15 x l7-nln., soft swelling with a chapped, weeping surface. At 19 days, the swelling was 10 m. in diameter and had a central ulcer 2 m. in diameter from which purulent material was draining. This swelling was still 10 m. in diameter with central dry eschar at both 32 days and 43 days after inoculation. By 62 days after inoculation the swelling had disappeared and the lesion had healed. 50 Necrgpsy findings (82 days after inoculation). No gross lesions were found . Histggtholgic findiggs. A granulana with central caseation necrosis surrounded by a few lymphocytes and macrophages and a moderate amount of connective tissue was found in the left prescapular lymph node (Figure 11). Also scattered, unencapsulated granulomas consisting of macrophages and giant cells were found. Only acanthosis, hyperkeratosis and dermal thickening with connective tissue were found in the skin at the site of inoculat ion . Fig 5-5 (Pen-mate of animals inoculated with M. aviun, laboratory strain) Clinical observations. No abnormalities were detected. Necrgpsy findiggs (82 days after inoculation). No gross lesions were found . Histogtholgic findiggs. No microscopic lesions were found. Fig 5-2 Clinical observations. Eight days after inoculation there was a swelling 14 m. in diameter at the site of inoculation. Fourteen days after inoculation a 16 x 20-nn.. soft swelling was found. No change was noted when the lesion was examined 19 days after inoculation. At 32 days after inoculation the swelling was soft and 15 an. in diameter with a central, Z-m. ulcer covered by an eschar. Forty-three days after inoculation the swelling was 20 mm. in diameter and the ulcer 7 mm. in diameter. Sixty-two days after inoculation the lesion was a 5 x 6-nln., 51 Figure 11. A granuloma found in the left prescapular lymph node of Fig 5-3 which was inoculated intradermally with M. avium, laboratory strain. Note central caseation necrosis (1) surrounded by macrophages, lymphocytes and a moderate amount of connective tissue (2). New Fuchsin - H & E. 1:75. 52 healing ulcer. Seventy-one days after inoculation the lesion was healed. Necrgpsz findings (138 days after inoculation). A 15 x lS-m., slightly thickened area was found in the skin. The distal 60 m. of the spleen was bluish-red. A tumorous mass approximately 30 x 20 x 10 m. was attached to the tip of the spleen. The central part of this mass was deep red. Histgathologic findings. A granuloma consisting of a group of macrOphages and lymphocytes was found in the left prescapular lymph node. The lesion was poorly encapsulated. Hyperkeratos is, parakeratosis, acanthosis and dermal thickening with connective tissue were found in the skin at the site of inoculation. Two large hematocysts filled with ghost cells and in process of organization were found in the section of the 8p leen . Pig 5-5 (Pen-mate of animals inoculated with M. gyium, laboratory strain) Clinical obsegations. No abnormalities were detected. Necrgsy finding (138 days after inoculation of pen-mates). One yellow, caseous lesion approximately 10 an. in diameter and a similar lesion approximately 5 mm. in diameter were found in the mesenteric lymph rides e Histgpatholgic findiggs. Two areas containing numerous granulomas consisting of macrOphages and/or giant cells were found in the parotid lynmh nodes. A few, similar granulomas were found in the submaxillary 53 lymph nodes. Two areas containing numerous scattered lesions ranging from unencapsulated granulomas made up of a few macrophages to large, coalescing, and well-encapsulated granulomas with caseous or caseocal- careous centers surrounded by macrOphages were found in the sections of the mesenteric lymph nodes. Pig 5-1 Clinical observations. Eight days after inoculation there was a swelling 10 m. in diameter at the site of inoculation. Fourteen days after inoculation the swelling was soft and 13 x17 m. At 19 days the lesion was a 15~x lS-m., soft swelling. No change was noted at 32 days. Forty-three days after inoculation the swelling was 15 m. in diameter and cmtained a central ulcer 10 m. in diameter. Sixty-two days after inoculation there was a slight swelling, 10 m. in diameter, with an 8-mm.-diameter, healing ulcer in the center. Seventy-one days after inoculation the ulcer was almost healed. Necroggz findiggs (215 days after inoculation). No gross lesions were found. Histgthologic findings. (he granulana with a caseocalcareous center surrounded by macrophages was found in the mesenteric lymph node. This was well-encapsulated. Hyperkeratosis, some acanthosis, and dermal thickening with connective tissue were noted in the area of inoculation. 54 Fig 5-6 (Pen-mate of animals inoculated with M. avium, laboratory strain) Clinical obsergations. No abnormalities were detected. Necropgy findings (215 days after inoculation). Three caseous lesions, approximately 2 m. in diameter, were found in l mesenteric lymph node . Histgatholggic findings. One unencapsulated granuloma consisting of a few giant cells and macrophages was found in the retropharyngeal lymph node. In the mesenteric lymph node, several granulomas with caseo- calcareous centers surrounded by a few macrOphages were found (Figure 12). These granulomas were well-encapsulated. Two granulomas were found in the section of the gastric lymph node. Each was made up of a few giant cells and macrophages and was unencapsulated. Culture 206-2, M. avium, swine origin Pig 10-3 Clinical observations. Seven days after inoculation there was a hard swelling 15 m. in diameter at the inoculation site. At 14 days after inoculation the area was unchanged but the swelling was red and fluctuating. Twenty-two days after inoculation the lesion was soft, fluctuating, denuded and 24 mm. in diameter. Twenty-nine days after inoculation a soft, ulcerated, 30 x 40-m. swelling was found. Thirty- six days after inoculation a soft, 30 x 35-m. swelling with a 10m.- diameter ulcer was found. 55 Figure 12. lbsenteric lymph node fran Pig 5-6, inocu- lated intradermally with g. aviun, laboratory strain. Note calcification (l), caseation (2),and area of ma’crophages and lymphocytes (3). New Fuchsin - H 6: E. x187. 56 Necropsy findings (43 days after inoculation). A swelling approxi- mately 25 m. in diameter with a central ulcer approximately 8 m. in diameter was found in the skin at the inoculation site. Gaseous foci, l m. in diameter, were found in the left prescapular lymph node. Gaseous foci, l to 10 m. in diameter, were found in the anterior mediastinal lymph nodes. Caseous foci up to 15 m. in diameter were ' found in the lungs. Histgpatholgic findiggg. Numerous granulomas were found scattered throughout the left prescapular lymph node. These ranged from small, unencapsulated groups of macrOphages and/or giant cells to well-encapsulated granulomas with caseocalcareous centers surrounded by macrophages. One area containing a few unencapsulated granulomas was found in the mesen- ' teric lymph node. These granulomas consisted of small groups of giant cells and macrophages. Numerous large granulomas were found in the anterior mediastinal lymph nodes. In the liver mmerous scattered granu- lomas with caseocalcareous centers surrounded by giant cells and macro- phages were found. These lesions were well-encapsulated. In the lungs granulomas with caseocalcareous centers surrounded by macrophages, giant cells, and lymphocytes were found. Daughter tubercles were present; 0 however , a 11 were we ll-encaps ulated . Pig lO-l Clinical observgtions. Seven days after inoculation there was a hard, 10 x 15m. swelling at the site of inoculation. No change was noted by 14 days after inoculation. By 22 days after inoculation the 57 swelling was soft, fluctuating, and measured 14 x 18 mm. Twenty-nine days after inoculation there was a 15 x 20-m., soft, fluctuating swelling at the'inoculation site. Thirty-six days after inoculation the lesion was lO‘x 30 mm., soft, and fluctuating. Forty-nine days after inoculation there was a soft, fluctuating, ulcerated swelling 15 x 30 mm. at the inoculation site. Necrgpsz findings (57 days after inoculation). One of the lateral retropharyngeal lymph nodes contained multiple, grayish-yellow foci 0.5 to 2 mm. in diameter scattered uniformly thr0ughout the node. A.7~mm. red area was found at the inoculation site. There was no ulcer. Histgpgtholggic findings. One unencapsulated granulome consisting of a few giant cells and macrophages was found in 1 section of a mesen- teric lymph node. A few unencapsulated granulomas consisting of a few macrophages surrounding a small group of necrotic cells were found in the bronchial lymph nodes. Numerous scattered lesions ranging from rather large, unencapsulated groups of macrophages to well-encapsulated granulomas with caseocalcareous centers surrounded by macrophages were found in the lateral retropharyngeal lymph nodes GFigures 13 and 14). Granulomas made up of small, unencapsulated groups of macrophages and/or giant cells were found in the left prescapular lymph node. The dermis was greatly thickened with connective tissue at the inoculation site. A few granulomas with caseous centers surrounded by macrophages and lymphocytes were found in the dermis. e '4’. . V, eu‘ '1‘ "TE-Vii e '0. Figure 13. Unencapsulated group of macrophages found in the lateral retrOpharyngeal lymph node of Fig 10-1, which was inoculated intradermally with Culture 206-2, M. avium, swine origin. New Fuchsin - H & E. x187. Figure 14. Well-encapsulated granulomas with caseocal- careous centers found in the lateral retropharyngeal lymph node of Pig lO-l, which was inoculated intradermally with Culture 206-2, M. avium, swine origin. New Fuchsin - H & E. x75. 59 Pig 10-& Clinical observations. Seven days after inoculation a hard, lO-mm.- diameter swelling was found at the inoculation site. Fourteen days after inoculation the swelling was similarly hard and 7 mm. in diameter. At 22 days after inoculation there was a 10~mm.-diameter swelling with a central 5-mm. ulcer present. Twenty-nine days after inoculation there was a lO-mm.-diameter swelling while the ulcer appeared to be healing. By 49 days the swelling was 5 mm. in diameter and the ulcer was healed. Necropsy findingg (113 days after inoculation). No gross lesions were found. Histgpatholggic findings. Scattered lesions ranging from small groups of macrOphages with no encapsulation to moderately encapsulated granulomas with caseous centers surrounded by macrophages were found in the mesenteric lymph nodes. Pig 10-2 Clinical observations. Seven days after inoculation there was a slight swelling 5 mm. in diameter found at the inoculation site. Four- teen days after inoculation, no swelling was detected. By 22 days, there was a hard, l4-mm.-diameter swelling at the site. The lesion was a soft, fluctuating swelling, 10 mm. in diameter, 29 days after inoculation. No change was noted at 36 or 49 days after inoculation. Necrgpsy findiggs (113 days after inoculation). A 6-mm.-diameter caseocalcareous focus was found in the center of the left prescapular 60 lymph node. At the inoculation site a 15 x Z-m. scar was found on the surface. On cross section, a 4 x 4 x 8-mm. lesion containing yellowish- green pus was found in the dermis. Histgpatholggic findings. Nunerous, scattered granulonnas were found in the left prescapular lymph node. One was well-encapsulated and had a caseocalcareous center surrounded by macrophages and a few lympho- cytes. This was surrounded by numerous daughter tubercles, sane with necrotic centers and others made up of giant cells and/or macrophages only. These daughter tubercles were either nonencapsulated or only slightly encapsulated. One large granuloma with a caseocalcareous center which was surrounded by a few smaller granulomas was found in the dermis. Lymphocytes and macrophages were found in these granulanas. All were we ll-encapsulated. Pig 10-5 Clinical observations. Seven days after inoculation the lesion at the inoculation site was a slight swelling 5 m. in diameter. _ Fourteen days after inoculation there was a hard swelling, 10 m. in diameter, found at the inoculation site. By 22 days after inoculation the lesion was a hard swelling, 18 m. in diameter. A similar lesion, 14 mm. in diameter, was found 29 days after inoculation. Thirty-six days after inoculation a soft swelling 10 m. in diameter was found. By 49 days after inoculation a lO-mm.-diameter, hard swelling was found which con- tained an ulcer 3 m. in diameter. 61 Necrogz findings (175 days after inoculation). No gross lesions were found. Histfltholggic finding. No microscOpic lesions were found. Pig 10-6 Clinical observations. No swelling was found at the inoculation site until 36 days after inoculation when a hard lO-m.-diameter swelling was found. Forty-nine days after inoculationn the lesion was a soft, fluctuating swelling, 7 m. in diameter. Necrgpsy findings (175 days after inoculation). Two caseocalcareous lesions, 1 approximately 10 x 10 x 2 m. and the other approximately 3 m. in diameter, were found in l submaxillary lymph node. Histgpgtholggic findings. Numerous scattered lesions were found throughout a submaxillary lymph node. These ranged fran small, unencapsu- lated groups of macrophages and/or giant cells to well-encapsulated granulomas with caseocalcareous centers. In these lesions, many macro- phages and few giant cells were found. Fig 10-2 (Pen-mate of animals inoculated with _1_i_. avium, swine origin) Clinical observations. No abnormalities were detected. Necrogz finding (182 days after inoculation). No gross lesions were found. Histmtholggic findings. No microscOpic lesions were found. 62 Fig 10-8 (Pen-mate of animals inoculated with M. avium, swine origin) Clinical observations. No abnormalities were detected. Necrgpsy findingg (182 days after inoculation). No gross lesions were found. Histgpatholggic findings. No macroscopic lesions were found. Culture 15D, Group III, pen origin Pig 161-3 Clinical observations. No lesions were detected at the inoculation site when examined at 7, 12, 19, 33, and 48 days after inoculation. Necropgy findings (138 days after inoculation). A slight thickening approximately 10 mm. in diameter was found at the inoculation site. Histgpatholggic findingg. There was some acanthosis, hyperkeratosis and slight dermal thickening with connective tissue at the inoculation site. Fig 161-6 Clinical observations. No abnormalities were detected. Necrgpsy findiggs. This animal was not necropsied. When slaughtered approximately 145 days after inoculation, no gross lesions were detected. 63 Culture 192W, Group III, pen origin Pig 161-5 Clinical observations. A swelling 10 x 15 mm. was found at the inoculation site 7 days after inoculation. No abnormalities were detected when the lesion was examined at 12, 19, 33, and 48 days after inoculation. Necrgpsy findings (138 days after inoculation). No gross lesions were found. Histmtholggic findings. No microscOpic lesions were found. Pig 161-2 Clinical obsegzations. A 6-nnn.-diameter swelling was noted 7 days after inoculation at the inoculation site. No abnormalities were found when the site was examined at 12, 19, 33, and 48 days after inoculation. Necropsz findings. This animal was not necropsied. No gross lesions were observed when slaughtered approximately 145 days after inoculation. Pig l6l-7 (Pen-mate of animals inoculated with Culture 15D or 192W) Clinicgl obserzgtions. No abnormalities were detected. Necrogz findgng (approximately 145 days after inoculation). This animal was not necropsied. No gross lesions were observed at slaughter. Pig l6l-§ (Pen-mate of animals inoculated with Culture 15D or 192W) Clinical observations. No abnormalities were detected. 64 Necrgsz findiggs (approximately 145 days after inoculation). This animal was not necrOpsied. No gross lesions were observed at slaughter. Pig 161-9 (Pen-mate of animals inoculated with Culture 15D or 192V!) Clinical observations. No abnormalities were detected. Necrgpsz findiggs (approximately 145 days after inoculation). This animal was not necropsied. No gross lesions were observed at slaughter. 1;. Oral Administration Culture 81-0, g. bovis, swine origin Fig 7-1 Clinngl observatifl. No abnormalities were detected. Necrogz findings (79 days after administration of culture). The right lateral retropharyngeal lymph node was approximately 1/3 filled with yellowish caseous material. The right submaxillary lymph node was en- larged (70 x 50 x 30 In.) and filled with yellow caseous material. Two lymph nodes were found in the area normally occupied by the left submaxil- lary lymph node. One of these nodes was 80 x 80 x 40 mm. and the other was 30 x 20 x 15 m. The nodes were filled with a greenish, tenacious material and had some firm areas filled with yellow caseous material. The anterior and posterior mediastinal and the bronchial lymph nodes were enlarged and conntained l- to 2-sIn.-diameter yellow foci scattered through- out. Several nodes from the gastric and hepatic group contained yellowish- white, caseous foci. Most of the mesenteric lymph nodes were filled with 65 yellowish-white, caseous foci. A lO-mm.-diameter, dull, whitish area was found in l kidney. Histgatholgic findings. The submaxillary lymph nodes were almost filled with lesions. In general, these were large granulcmnas with caseous or caseocalcareous centers surrounded by macrophages and were well-encapsulated. Also numerous small granulomas made up of small groups of macrophages or macrOphages and giant cells were found. Several small granulomas consisting of a few macrophages and/or giant cells were found in the left medial retropharyngeal lymph node. Numerous scattered and confluent lesions ranging from small groups of macrophages and giant cells with no encapsulation to large, well-encapsulated granulanas with caseo- calcareous centers surrounded by macrophages were found in the gastric, hepatic, and right lateral retropharyngeal lymph nodes. Similar lesions were found in the mesenteric lymph nodes except that they were still more numerous. In some nodes the granulomas had coalesced until the resulting lesion almost filled the node. Several focal granulomas, some of which were becoming confluent, were found in the left papliteal lymph node. Early, central areas of caseation necrosis surrounded by macrophages were found in these lesions. They were unencapsulated to moderately encapsulated. Lesions similar to those found in the submaxillary lymph node were also found in the right prescapular lymph node. In the kidney a few small, scattered, unencapsulated granulomas consisting of a few macrophages surrounded by lynphocytes were found. Also there were numerous small areas of lymphocyt ic infiltration. One granuloma with early central caseation necrosis surrounded by macrophages and lymphocytes 66 was found in an interlobular area of the liver. Also, isolated areas of leukocytic infiltration were found within and beside the lobules. Fig 7-6 (Pen-mate of animals fed Culture 81-0, M. bovis, swine origin) Clinical observations. No abnormalities were detected. Necrgsz finding (79 days after administration of culture to pen-mates). Several l- to Zulu-diameter, yellowish foci were found in the gastric lymph nodes. A caseous, yellow lesion approximately 5 x 10 x 10 Inn. was found in l mesenteric lymph node. Histgatholggic findiggs. A few granulanas were found near the capsule of the left parotid lymph node. These consisted of areas of early central caseation necrosis surrounded by macrOphages and a few giant cells. Occasionally noncaseous granulomas were found. Scattered focal or dif- fuse, unencapsulated lesions consisting of large groups of macrophages and giant cells were found in the right submaxillary lymph node. Early caseation necrosis was sometimes found. Several unencapsulated to moderately encapsulated granulomas were found in the bronchial lymph nodes. Some had small caseous centers, while others consisted of small groups of macrOphages. Nunerous scattered granulomas with little or no encapsulation were found in the gastric and hepatic lymph nodes. Some had caseous or caseocalcareous centers, but the lesions were mainly groups of macrophages and few giant cells. In the area in which the gross lesion of the mesenteric lymph node was found numerous, discrete or con- fluent granulomas, some with a moderate amount of central, caseous or caseocalcareous material were found. The macrophage was the predominant 67 cell in these lesims. Some of the granulomas were moderately encapsulated while others were not. Other areas of the mesenteric lymph nodes were normal. In the lung, 1 unencapsulated peribronchial granuloma consisting of macrophages, lymphocytes, and plasma cellshwas found. Many of the bronchi contained macrOphages. The alveolar walls were thickened. Fig 7-2 Clinical observgt ions. No abnormalities were detected. Necrogz finding (134 days after administration of the culture). The left submaxillary lymph node was enlarged (70 x 40 x 30 m.) and filled with yellow caseous or caseocalcareous, discrete or confluent lesions. The right submaxillary lymph node was enlarged (50 x 40 x 20 nan.) and contained similar lesions. The right bronchial lymph node was enlarged (50 x 20 x 25 um.) and filled with yellowish, caseocalcareous lesions from 1 to 5 m. in diameter. The left bronchial lymph node con- tained a few scattered, yellowish, caseous lesions 1 to 3 m. in diameter. One of the anterior mediastinal lymph nodes contained a yellowish, caseous lesion approximately 5 m. in diameter. Yellowish, caseous lesions from 3 to 5 nun. in diameter were found in the gastric and hepatic lymph nodes. Scattered, caseous lesions 1 to 3 m. in diameter were found throughout the mesenteric lymph nodes. Scattered, yellowish-white foci up to approxi- mtely 0.5 m. in diameter were found in the liver. Histogtholggic findiggs. Numerous, scattered granulomas were found throughout the bronchial and the anterior mediastinal lymph nodes. Many of these granulomas appeared to be coalescing. Most had caseous centers; 68 some also contained calcium. Small granulomas without necrotic centers were also found. These granulomas were made up of macrophages, giant cells, and lymphocytes. In general, the lesions were well-encapsulated, but some had little or no encapsulation. Sinilar lesions were found in the submaxillary lymph nodes, except that more coalescence, necrosis, calcification, and encapsulation were present. Daughter tubercles were also seen. In an area of the left parotid lymph node, several scattered granulomas were found. Most had caseous or caseocalcareous centers surrounded by macrophages. Early attempts at encapsulation were found. Nunnerous granulomas were found in the mesenteric lymph nodes. Most had caseous or caseocalcareous centers surrounded by macrophages. Encapsula- tion varied from none to extensive. The lesions in the gastric lymph nodes were numerous and scattered, ranging from small granulomas without necrotic centers to moderate-sized granulomas with caseous or caseocal- careous centers. MacrOphages and a few lymphocytes were found in these lesions. The encapsulation varied from none to extensive. Similar lesions were found in the hepatic lymph nodes except that, in general, there was less encapsulation present. In the liver, occasional areas of lympho- cytic infiltration were found. These were not granulunas. Numerous granulomas consisting of groups of macrophages and lymphocytes were found in the kidneys. In the lung, occasional granulomas consisting of accumu- lations of lymphocytes, macrophages, and giant cells were found. Pig 7-§ (Pen-mate of animals fed Culture 81-0, M. bovis, swine origin) Clinical observations. No abnormalities were detected. 69 Necrgpsy finding (134 days after administration of cultures to pen-mates). No gross lesions were found. Histggatholgic findings. No microscoPic lesions were found. Pig 7"; Clinical observations. No abnormalities were detected. Necropsy finding (203 days after administration of organisms). Several scattered calcareous foci, l to 10 m. in diameter, were found in the left parotid, right submaxillary, right medial retropharyngeal, bronchial, anterior mediastinal, gastric, and mesenteric lymph nodes. A l-mm-diameter caseous nodule was found in the cortex of the left pre- scapular lymph node. A few 1- to 3m.-diameter, whitish foci were found scattered throughout the spleen. Histgatholggic findigg. The submaxillary lymph nodes were almost filled with discrete or confluent granulomas. Most had extensive caseo- calcareous centers surrounded by a few macrophages. In addition, small, unencapsulated granulomas consisting of groups of macrophages were found. Similar lesions were found in the right medial retropharyngeal and left parotid lymph nodes. Nunerous granulanas were found almost filling the right bronchial lymph node. Many were becoming confluent. Nunerous daughter tubercles were found. Encapsulation varied from slight to exten- sive. Similar lesions were found in the left bronchial and anterior mediastinal lymph nodes, except that the encapsulatian was more pronounced. Nnnerous scattered lesions ranging from small groups of macrophages to 70 large confluent granulomas with caseocalcareous centers were found in the mesenteric lymph nodes. Encapsulation ranged from none to extensive. Similar lesions were found in the gastric lymph nodes. Occasional granu- lomas consisting of l to 2 giant cells and a. few macrophages were found in the left prescapular lymph node. In the spleen, a large granulana with caseocalcareous center» and extensive encapsulation was found. Fig 7-4 (Pen-mate of animals fed M. bovis, swine origin) Clinical observations. No abnormalities were detected. Necrgsg findings (203 days after organisms were administered to pen-mates). The submaxillary lymph nodes were greatly enlarged and filled with confluent or discrete yellow caseocalcareous lesions. The bronchial, anterior and posterior mediastinal, and gastric lymph nodes contained numerous, scattered, discrete or confluent, yellowish foci l to 4 sun. in diameter. In the liver, scattered white areas 4 to 5 m. in diameter were found lying just under the capsule. Histgatholggic findings. The submaxillary lymph nodes were almost filled with lesions ranging from small, unencapsulated granulomas made up of groups of macrophages to very large, confluent, well-encapsulated granu- lomas with caseocalcareous centers. In the parotid lymph nodes, numerous, scattered, unencapsulated granulanas made up of groups of giant cells and macrophages were found. Numerous, small granulanas, some having caseocalcareous centers and being slightly encapsulated and others having little or no encapsulation nor necrosis, were found in the lateral retro- pharyngeal lymph nodes. Nunerous scattered lesions ranging from small 71 unencapsulated groups of macrophages to encapsulated granulomas with caseocalcareous centers were found in the medial retropharyngeal and anterior mediastinal lymph nodes. Similar lesions were found in the bronchial and posterior mediastinal lymph nodes, except that giant cells were more prevalent in these lesions. One area containing several granu- lomas was found in the mesenteric lymph nodes. These granulomas ranged from unencapsulated granulomas made up of small groups of giant cells and macrOphages to slightly encapsulated groups of macrophages with early, central caseation necrosis. Similar lesions were found almost filling the gastric lymph nodes. A.noncaseating granuloma consisting of a small group of giant cells and macrOphages was found in the left pre- scapular lymph node. A group of granulomas, some with early caseation necrosis in their centers, were found in the liver. Also, scattered throughout the parenchyma of the liver were small areas of lymphocytes and/or macrophages. In the lung, 1 small granuloma‘was found which con- sisted only of a few macrophages. There was also alveolar hemorrhage and thickening of the alveolar wall. Culture 172C1-l, Group III, swine origin Pig 6-1 'Clinical observations. No abnormalities were detected. Necrgpsy findingg (78 days after adndnistration of the organisms). Whitish foci, 2 to 3 mm. in diameter, were found scattered near the sur- face of the liver. 72 Histgggtholgic finding. Many giant cells were found scattered throughout the mesenteric lymph nodes. These usually occurred singly, lying in groups of reticulum cells, but occasionally they were surrounded by groups of macrophages. Also granulanas with caseous or caseocalcareous centers surrounded by macrophages were found. fine was well-encapsulated while the others were not encapsulated. Pig 6-6 (Pen-mate of animals fed Culture 172C1-l, Group III, swine origin) Clinicgl observations. No abnormalities were detected. Necrgsz finding (78 days after administration of the organisms). No gross lesions were found. Histggtholggic findings. No microscopic lesions were found. Fig 6-2 Clinical obseggations. No abnormalities were detected. Necrgpsg finding (136 days after administration of the organisms). Several yellowish, caseous or caseocalcareous lesions were found in 2 of the mesenteric lymph nodes. Histopatholgic findings. Numerous, scattered granulomas were found in the mesenteric lymph nodes. Most of these granulomas‘were well- encapsulated and had caseocalcareous centers surrounded by macrophages and lymphocytes. A few had little or no necrotic material or encapsula- tion and were made up of macrophages and lymphocytes. 73 Pig 6-5 (Pan-mate of animals fed Culture 17201-1, Group III, swine origin) Clinical observations. No abnormalities were detected. Necrgpsz findings (136 days after administration of the organisms). No gross lesions were found. Histgpatholggic findings. One well-encapsulated granuloma, consist- ing of macrophages surrounding a few necrotic neutrophils, was found in the right submaxillary lymph node. Several lesions ranging from unencapsu- lated groups of macrophages to well-encapsulated granulomas with caseocal- careous centers were found in the mesenteric lymph nodes. Numerous scattered granulomas, consisting of groups of giant cells and macrophages, ‘were found in the medial retropharyngeal lymph nodes. Pig 6-3 Clinical observations. No abnormalities were detected. Necrgpsy findingg (206 days after administration of culture). Numerous caseous foci, 2 to 10 mm. in diameter, were present throughout the length of the chain of mesenteric lymph nodes. Similar lesions were found in the gastric and hepatic lymph nodes. Numerous caseous foci, 0.5 to 4 mm. in diameter, were found in the submaxillary lymph nodes. Histgpgtholggic findingg.‘ Several scattered granulomas with encapsu- lation ranging from moderate to none were found in the submaxillary lymph ‘nodes. Some of these granulomas had caseocalcareous centers, while others ‘were made up of macrOphages only (Figure 15). Numerous scattered granulomas 74 were found throughout the mesenteric, gastric and hepatic lymph nodes CFigure 16). These ranged from unencapsulated groups of macrophages to large, well-encapsulated granulomas with caseocalcareous centers. Giant cells were prevalent in these lesions. Similar lesions were found in the colic lymph nodes. Pig 6-& (Pan-mate of animals fed Culture 17201-1, Group III, swine origin) Clinical obseggations. No abnormalities were detected. Necrgpsy finding; (206 days after administration of the culture). Two calcified foci, 1 mm. in diameter, were found in the mesenteric lymph nodes. Histgpgtholggic findingg. One granuloma consisting of an area of coagulation necrosis surrounded by macrophages was found in the left sub- maxillary lymph node. Pour unencapsulated granulomas were found in the right bronchial lymph node. Three of these consisted of small groups of macrophages and/or giant cells. The other had a small caseous center sur- rounded by macrophages. Numerous scattered lesions ranging from small groups of macrophages and/or giant cells to granulomas with caseocalcareous centers were found in the mesenteric lymph nodes. Encapsulation was slight to absent. Numerous, scattered granulomas consisting of groups of macro- phages and giant cells were found in the left prescapular lymph node. These lesions were unencapsulated. 75 n n A" . tr: . - - .n - ~t.‘ " , {\3 "fig“ "C 9‘ II . ‘ *4. $43' . Est-‘5“ w .1. §:1§€§k*§ ‘, . .Is'V‘. \ ‘ -. I ‘ ‘1 ‘- a .- t, ‘f Figure 15. Two contiguous granulomas found in the left submaxillary lymph node of Pig 6-3 to which Culture 172C1-1, Group-III mycobacteria, swine origin were administered orally. Note the moderately encapsulated granuloma with caseocalcareous center (1) and the adjacent unencapsulated, noncaseous granulomas (2). New Fuchsin - H & E. :75. Figure 16. An unencapsulated, noncaseaEing granuloma found in a mesenteric lymph node of Pig 6-3 to which Culture 1720 -l, Group-III mycobacteria, swine origin was administered oral y. New Fuchsin - H & E. 3187. 76 fl. avium, laboratory strain Pi - Clinical observations. No abnormalities were detected. Necrgpsx finding (52 days after administration of organisms). No gross lesions were found. Histgpathologic finding. No microscOpic lesions were found. Pig 8-1 (Pen-mate of animals fed 14. avium, laboratory strain) Clinical observations. No abnormalities were detected. Necrgpsz finding (52 days after inoculation). No gross lesions were found . gistopatholgic findings. No microscopic lesions were found. Pig 8-3 Clinical observations. No abnormalities were detected. Necrgpsg findings (108 days after inoculation). No gross lesians were found. Histgpathologic findings. No microscOpic lesions were found. fig 8-§ (Pen-mate of animals fed y. avium, laboratory strain) Clinical observations. No abnormalities were detected. Necrgpsg findings (108 days after administration of the culture). No gross lesions were found. 77 Histgpatholggic findiggs. No microscOpic lesions were found. Pig 8-& Clinical observations. No abnormalities were detected. Necrgsx findings (169 days after administration of organisms). No gross lesions were found. Histgpatholggic finding. 'l‘wo granulomas were found in the submaxil- lary lymph nodes. Both had caseocalcareous centers surrounded by macro- phages and were only slightly encapsulated. fig 8-6 (Pen-mate of animals fed g. avium, laboratory strain) Clinical observations. No abnormalities were detected. Necrgpsx findings (169 days after administration of organisms). No gross lesions were found. Histgatholgic findiggs. No microscOpic lesions were found. Culture 206-1, 2!. avium, swine origin Pig 9-2 Clinical observations. No abnormalities were detected. Necrgpsz findiggs (57 days after administration of organisms). No gross lesions were found. Histgatholgic finding. Numerous scattered granulomas were found in the mesenteric lymph nodes. These granulomas consisted of groups of 78 giant cells and/or macrophages. In some, there was early, central casea- tion necrosis. None was encapsulated. Pig 9-1 Clinical observations. No abnormalities were detected. Necrgpsz findings (57 days after administration of culture). No gross lesions were found. Histgpgtholggic findiggs. Numerous, scattered granulomas consisting of small groups of giant cells and macrophages were found in the sub- maxillary lymph nodes. These lesions were not encapsulated. Numerous granulomas were scattered throughout most of the mesenteric lymph nodes. For the most part, these consisted of accumulations of giant cells and macrophages with no encapsulations. However, some had caseous centers and were slightly encapsulated. Pig 9-3 Clinical observations. No abnormalities were detected. Necrgpsz findings (115 days after administration of culture). Circum- scribed, yellowish-green, caseous foci, l to 7 mm. in diameter, were found throughout the mesenteric, hepatic, and gastric lymph nodes. Histgpgtholggic findings. Numerous scattered lesions, ranging from nonencapsulated groups of macrophages to well-encapsulated granulomas with caseocalcareous centers surrounded by macrophages were found in the mesen- teric, hepatic, and gastric lymph nodes. 79 Pig 9-4 Clinical observations. No abnormalities were detected. Necropsy findings (115 days after administration of culture). Throughout the length of the mesenteric lymph nodes there were whitish- yellow, circumscribed, caseocalcareous foci, l to 2 mm. in diameter. Similar lesions were found in the gastric and hepatic lymph nodes. Histgpatholggic findings. Numerous, well-encapsulated granulomas with caseocalcareous centers were found in the gastric and hepatic lymph nodes. In addition, slightly encapsulated, daughter tubercles with or without caseous centers were found. Numerous, scattered lesions were found in all sections of the mesenteric lymph nodes. These ranged from unencapsulated groups of giant cells and/or macrophages to large, well- encapsulated granulomas with caseocalcareous centers surrounded by macro- phages which almost filled the node. Numerous, scattered granulomas con- sisting of a few giant cells and/or macrOphages and no encapsulation were found in the right submaxillary lymph node. In the liver, a few, scattered, unencapsulated granulomas were found in the parenchyma. These 'were made up of a few giant cells and macrOphages. Pig 9'5 Clinical observations. No abnormalities were detected. Nécrogsz findings (171 days after administration of culture). About 301 of the mesenteric lymph nodes contained circumscribed yellowish caseo- calcareous foci, approximately 2 to 5 mm. in diameter. 80 Histgggthologic findings. A few unencapsulated granulomas con- sisting of a few giant cells and/or macrOphages were found in the parotid lymph nodes. In the mesenteric lymph nodes, numerous, scattered lesions, ranging from unencapsulated groups of macrOphages to large coalescing, well-encapsulated granulomas with central caseation necrosis were found. In general, the encapsulation was not extensive. Pig 9-6 Clinical observations. No abnormalities were detected. Necrgpsz findings (171 days after administration of the culture). Approximately 15% of the mesenteric lymph nodes contained yellowish, caseous foci, 2 to 4 mm. in diameter, in the cortical area. Histgpgtholggic findings. One well-encapsulated granuloma with caseous center surrounded by a few macrophages was found in a submaxil- lary lymph node. Numerous, scattered lesions were found throughout the ‘mesenteric lymph nodes. They ranged from nonencapsulated groups of macro- phages to well-encapsulated granulomas made up of almost entirely central caseocalcareous material. In general, the lesions were not well- encapsulated. Pig 9-8 (Pen-mate of animals fed Culture 206-l, y. avium, swine origin) Clinical observations. No abnormalities were detected. Necrgpsz findiggs (177 days after administration of culture). A.few circumscribed, yellowish, caseous foci, 2 to 4 mm. in diameter, were found in the submaxillary lymph nodes adjacent to the capsule. In the 81 liver, small, yellowishdwhite, solid foci, approximately 3 mm. in diame- ter, were found immediately beneath the capsule. Histgpatholggic findings. Numerous, scattered granulomas were found in the submaxillary lymph nodes. These ranged from groups of macrophages with no necrosis or encapsulation to granulomas with caseocalcareous centers surrounded by macrOphages and a moderate amount of connective tissue. In the liver, areas containing several lymph follicles each were found extending into the parenchyma from the capsule. These lesions were not considered granulomatous. Pig 9-Z (Pen-mate of animals fed Culture 206-1, 5. aviun, swine origin) Clinical observations. No abnormalities were detected. Necrgpsz findings (177 days after administration of the culture). Approximately 3% of the mesenteric lymph nodes contained circumscribed, yellowish, caseous foci, 2 to 10 mm. in diameter. Histopathologic findings. Numerous, scattered lesions were found in the mesenteric lymph nodes. These ranged from a few which were made up of groups of macrOphages and/or giant cells and were not encapsulated to well-encapsulated granulomas with caseocalcareous centers which appeared to be coalescing. uninoculated Control Agimals Uninoculated animals served as controls for each phase of this experi- ment. In all, 15 animals were used for this purpose. 82 No clinical signs of disease were observed in any of the animals. None of the antmals had any reaponse to avian or mammalian tuberculin. Post~mortem examinations were performed on 3 of these animals and no gross or microsc0pic lesions were found. The remaining animals were slaughtered in order to salvage their meat value. 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