THE LESIONS RESULTING FROM INOCULATION OF CALVES WITH ATYPICAL MYCOB‘ACTERIA Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY M. D. McGavin 1964 jHESIS This is to certify that the thesis entitled THE LESIONS RESULTING FRO! INOCULATIW OF CALVES WITH ATYPICAL MYCOBACTERIA presented by M. D. McGavin has been accepted towards fulfillment of the requirements for 6,762 WZMJZ Major professor Date May 22 , 1964 0-169 ABSTRACT THE LESIONS RESULTING FROM INOCULATION OF CALVES wrm AHPICAL MICOBACTERIA By M. D. McGevin Fifty-five celves of verious breeds end both sexes. end between six end ten months of ego. were inoculeted with nycobecterie. tech celt‘ received e single culture. Unless otherwise specified. calves were inoculated with either 1 mg. or 2.2 ng.. wet weight. of orgenisne intre- dernelly on the legs. They were tuberculin-tested on the caudal told with ne-elien tuberculin. by the conperetive cervicel nethod using evien end nenelien tuberculins end Johnin, end exenined by necropsy 8-12 weeks efter inoculetion. Thirty-seven celves were iinoculeted with Runyon Group III swoobecterie. 11 with Group IV woobecterie. 2 with g. m. l with g. 5533 end 1 eech with killed cultures of l!- m. y. m. e Group III of bovine origin end e Group IV swoobecteriul. Four of seven Group III cultures of bovine origin. inoculeted into 11+ celves. produced either e prinery couple: or generelized diseese in 8 celves. Two other cultures teiled to produce e printer-y complex end enother produced this in only one of three celves .' ‘lhe six different Group III cultures of swine origin were ell isoleted from swine nesenteric lynph nodes end were inoculeted into 11 calves. Only one culture produced e prinery complex with grenulones et the inoculetion site end in the left prescepuler lymph node. M. D. McGavin or seven calves inoculated with a total of four cultures of ”pseudo- chromes". five had no lesions and two had only small grenulomas at the inoculation sites. Four of these calves had each received a total of 10 lg.. wet weight. of organisms. 2 mg. being administered by each of the peroral. subcutaneous. intredermal. intramuscular end intreperitoneal routes. Five calves inoculated with three cultures of Group III nycobacteria of soil or cattle feed origin did not develop primary complexes. Six.cultures of Group IV myccbecteria were injected into 11 calves. # of which received a total of 10 ng.. administered by the perorel. subcu- taneous. intradermal. intreperitoneel end intramuscular routes. Four of these cultures were isolated from bovine "skin lesions”. Two of then caused no lesions in the experimental calves. One culture produced a slall intredermal grenulona in one calf. but no lesions in a second calf receiving the 10 ng. dose. The fourth culture produced no lesions in one calf. but the calf which received 10 mg. had encapsulated nonprogressive granulomas at the skin injection site end in the lymph nodes draining the intradermal. subcutaneous. end intramuscular injection sites. Both calves infected with 11. .bgyi; developed generalized disease. The calf injected with n. M developed only a nodule at the injection site. None of the calves injected.with killed cultures develOped lesions. In every case the comparative cervical test differentiated between those calves with progressive disease, as detormined by histological examination. and animals with either no lesions or localised nonprogressive lesions. Four animals inoculated with live mycobacteria end with no lesions. and one with e nonprogressive lesion. gave positive caudal fold tests. All M. D. McGavin calves inoculated with killed mycobecteria gave negative comparative cervical results. but one gave a positive caudal fold reaction. THE LESIONS RESULTING FROM INOCULATION OF CALYES WITH ATYPICAL MICOBACTERIA I 04 {.4‘ .\O 0 M.’ D.“'Mchvin A THESIS Submitted to .Michigen State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pathology 1964 47/1/7/ - I 22-L’2 AC KNOWWTS This work was carried out as part of a cooperative project between the Department of Microbiology and Public Health and the Department of Pathology. It has been a pleasure to work with this group. and the writer wishes to record his indebtedness to Dr.‘w. L. Hallmann for his stimulating discussions and advice and to Dr. V. H. Mallmann for her cooperation and supplying of the cultures used in the work. To Dr..C. C. Merrill. I would express my gratitude for offering me the opportunity to do this work and for his constructive criticisms of the manuscript. The tuberculin testing was carried out solely by Dr. James A. Rey end his readiness to come to my help when the work load became especially heavy will always be appreciated. Preparation of the histologic slides wee the work of Mrs. B. Kay Troeko. I. II. III. IV. TABLE OF CONTENTS INTRODUCTION AND OBJECTIVES . . . . . . . . . . . . . . mwummse O O O O O O O O O O O O O O O O O “mum AND mops O O O O O O O O O O O O O O O O O Experimentalinimels.................. -Inocu‘t1°n3eeeeeeeeeeeeeeeeeeeeee nocropsyTOChniqqueeeeeeeeeeeeeeeeee Bietopathologio Technique . . . . . . . . . . . . . . . Bacteriologic Technique . . . . . . . . . . . . . . . . Tuberculin Test Technique . . . . . . . . . . . . . . . Hematologic Technique . . . . . . . . . . . . . . . . . RESULTS . . . . . . . . . . . . . . . . . . . . . . . . Calves Inoculated with Group III Mycobacteria of BovinoOrigin..................... Calves Inoculated with Group III Mycobacterie of MOMgln....o...o............. Calves Inoculated with Group III.Hycobacteria of ‘FCCdorSOflorigineeeeeeeeeeeeeeeeee Calves Inoculated with Pseudochrome Hyeobacteria . . . . Calves Inoculated with Group IV Mycobacteria . . . . . . CalvesInoculetedwithg.§_v_i_ug . . CalvesInoculatedwithfi.bovis . . . . . . .. . . .. DISCUSSION Evaluation of Pathogenicity of Atypical Hycobecteria . . Relationship Between Ulceretion at the Skin Inoculation Site and Extent of Lesions . . . . . . . . . . . . . . . iii 14 15 17 19 22 22 23 26 26 6O 71 76 82 96 98 131 135 Page Evaluation of Tuberculin Sensitivity in Relation to “Sions hadnCOd O O O O O O O O O O O I O O O O O O O O O 138 Eva ation of Tuberculin Sensitivity Produced by Killodl‘lycobacteria_....................1'+2 SW 0 O O C O O O O O O O O O O O O O O O O O O O O 0 1n3 LIST OF mm O O O O O O O O O O O O 0 O O 0 O O O O 1’45 iv Table I. II. III. IV. V. VII. VIII. IX. X. 113T OF TABLES Index to inoculum. group. calf no. and page no. “3‘03“theeeeeeeeeeeeeeeeeeeeeee Smary of lesions and bacteriologic isolations from adv.‘ mocmted with WCOb‘Ctari‘ e e e e e e e e e e e I Tuberculin test results of calves infected with mywbactari‘eeeeeeeeeeeeeeeeeeeeee Results of hematologic examination of blood samples taken.immediete1y prior to necropsy . . . . . . . . . . . Summary of the development of skin lesions at the inoculation site in calves inoculated with Group III ”Nb‘cuflIOfmoori-gineeeeeeeeeeeeee Summary of the development of skin lesions at the inoculation site in calves inoculated with Group III ”Wb‘curi‘OIWOOTImeeeeeeee~eeeeeee Summary of the development of skin lesions at the inoculation site in calves inoculated with Group III mycobacteriaoffeedendeoilorigin .......... Summary of the development of skin lesions at the inocu- lation site in calves inoculated with pseudochromes . . . Summary of the development of skin lesions at the inocu- lation site in calves inoculated with Group IV mymbactoria...................... Summary of the development of skin lesions at the inocu- lation site in calves inoculated with 5. 9221.5. and 50m.OOOOOOOOOOOOOOOOOOOOOOO I Correlation of caudal fold tuberculin test results “lull-.810"eeeeeeeeeeeeeeeeeeeeee Correlation of comparative cervical tuberculin test resultswithlosions.................. Summary of results of tuberculin tests on calves injCCtd'iulkmOdIyCOb‘ctori‘e ee e e e ee e e e e Page 2b 107 117 122 125 127 128 129 129 130 Figure 13 1“ 15 LIST G FIGURES Left prescapuler lymph node from calf #3, inoculated with51C-O, aGroupIIInwcobaoterium. e e e e e e e e Left prescapuler lymph node from calf 1&3. inoculated with 516.0. .mewwb‘cunue e e e e e e e e Medial retropharyngeal lymph node from calf 1&3, inocu- latedwith 510-0. aGroupIIIwcobacterium . . . . . . Hesenteric lymph node from calf 1&3. inoculated with SIG-og‘GmupnIVCGb‘Gtmm eeeeeeeeeee Leptcmeninges adjacent to corpus striatum from calf 1&3. inoculated with 510-0. a Group III myoobaoterium . . . Leptomeninges adjacent to corpus strietum from calf b3. inoculated with 510-0. a Group III wcobacteritn . . . Left prescapuler lymph node from calf #5. inoculated fith680-O. ‘mp IIIwwb‘Ctmu-e e e e e e e e e Lungfromcalf 1&5. inoculated with 68C-0. aGroupIII ”commueeeeeeeeeeeeeeeeeeeee left prescapuler lymph node from calf 1+5. inoculated with680-O. ‘Gmnpxnwmb‘cuflue e e e e e e e e Left prescapuler lymph node from calf 1&5. inoculated Vii-I168“. ‘m‘qlp Invmb‘cuAMe e e e e e e e e Left prescapuler lymph node from calf b5. inoculated With 686—0. aGrOtmIIIwoObacteriul. e e e e e e e e Posterior mediastinal lymph node from self 1&5. inocula- td‘ith68c-O. ‘manmblcuriu‘e e e e e e e Posterior mediastinal lymph node from calf 1&5. inocula- tedwith680-0. aGroup IIIwcobacterium . . . . . . . Posterior mediastinal lymph node from calf its. inocula- tedwith680-O. aGroupIIIwoobacteriul e e e e e e e LiverfromcalfhvS. inoculated with 680-0. aGroup III WGOb‘CtOflmeeeeeeeeeeeeeeeeeeeee vi Page 35 35 1+2 1+2 1&7 1+7 Figure l6 l7 19 20 21 22 23 25. 26 27 28 29 30 Lung from calf 1&5. inoculated with 68C-0. a Group III Iwc0bacteriml....................o. Left prescapuler lymph node from calf 7. inoculated with 710-0. a Group III myoobacterium . . . . . . . . . Skin inoculation site from calf 52, inoculated With 117B-O.aGroupIVlwc0bacteriml. e e e e e e e e e e e Skin inoculation site from calf 52. inoculated with 117B-O.aGroupIVnchbacteriml.. ee e eee e e e e Right axillary lymph node from calf 51. inoculated with 7,-1. aGroupIVWOObacterittl.. ee e e e ee e e e e Right axillary lymph node from calf 51. inoculated with 7F-1.aGroupIVw00bactor1nn............. Internal iliac lymph node from calf 51. inoculated with 7,-1.CGPOQPIVWOOhctDr1ueeeeeeeeeeeee Internal iliac lymph node from calf 51. inoculated with ”-1. ‘GroanvwmbOCtCflu-e e e e e e e e e e e e e Right isohiatic lymph node from calf 51. inoculated with 7F-1. aGroup IV woobacterium . . . . . . . . . . Anterior mediastinal lymph node fra calf 20. inocula- mumflemeeeeeeeeeeeeeeeeeee Left prescapuler lymph node from calf 20. inoculated flth H. m 0 O O O O O O O O O O O O O O O O O O O 0 left prescapuler lymph node from calf 20. inoculated “mgomOOOOOOOOOOOOOOOOOOOOO Skin inoculation site from calf 20. inoculated with neMe~eeeeeeeeeeeeeeeeeeeeeee Skin inoculation site from calf 20. inoculated with BOMOOOOOOOOOOOOOOO000...... Skin inoculation site from calf 26. inoculated with killedn.m eeee‘eeeeeeeeeeeeeeee vii Page 86 88 91 91 92 92 103 103 104 101+ 105 105 I. INTRODUCTION AND OBJECTIVES This study was authorized under contract No. lZ-Ih-loo-h5ll (45) between the United States Department of Agriculture and.Michigen.State University. for investigations of the cause or causes of no-gross-lesion tuberculin reactors and to improve methods of diagnosis of bovine tubercu- losis. Item VI of amendment No. l of this contract states that the con- tractor shall (a) Artificially expose susceptible non-tuberculin reacting cattle from a tuberculosis-free herd or herds to virulent fit tghgrgnlggig,and/or antigenically related agents (b) Follow the development of the disease in arti- ficially exposed cattle through responses. such as. ... serologic and allergic response. post-mortem bacteriological examination of tissues as well as gross and microscopic tissue changes resulting from b‘mfl‘l mmue Verious atypical myccbaoteria had been isolated from.bovine and swine specimens and from inanimate sources. EXperimente were designed to shed light on the following problems; (a) the infectivity for calves of 0.1 and 1.0 mg..wet weight. of atypical swoobacteria and. H- m. (b) the patho- genicity of Group III mycobacteria of bovine. swine and feed and soil origins. of pseudochrome mycobacteria and.Group IV myoobacteria for calves. (c) the ability of killed mycobaoterie to induce sensitivity to tuberculosis. (d) the ability of pseudochrome and Group IV myccbecteria to induce.pathc- genie effects in calves using large doses by several routes of administra- tion. II. RIVIEW'OF LITERATURE Hycobecterie which did not conform to the description of the classi- cel.myoobecteria have been known for many years. Xalaberder (1961) wrote: Scarcely had the echoes of the report of Koch's dis- covery of the tubercle bacillus died awey than other mycobacteria were discovered that were unclassified in relation.to morphology or because they were non- pathogenic when tested in laboratory animals (Alveres and Tavel. 1885: Nocard and Roux. 1885). Iarlson (1958) has drawn attention to the taxonomic difficulties in classifying the myoobacteria.end states that up to 1905 there were 163 numbered.strains. Mycobacteria are widespread in nature and Frey and Began (1931) were able to demonstrate acid-fast bacteria in hundreds of soil samples from various parts of the U.S.A. Beceuse they are common in soil. myoobacteria are frequently found in vegetation and in the gastro- intestinal tracts of herbivorous animals. Iarlson (1958) points out that acid-fast bacteria have been found in laboratories. in distilled water tanks. table-top dust. rubber hoses attached to water taps and even in the water bottles used for preparing the Ziehleeelsen stein. In fact. Pinner (1935) has stated that "acid—fast bacilli have been isolated from almost any material that was properly.scrutinised." It can be appreciated. then. that contamination of biological specimens by such widespread.myco- bacteria could.lead to confusion and such nonpathogenic organisms would have to be distinguished from pathogenic myeobacteria when acid-fast organisms are isolated from animal tissues. For many years those acid- fast bacteria which grew rapidly and which did not produce progressive disease in the guinea pig were classified as ”saprOPhytes" and discarded 2 3 (Runyon. 1959). However. the repeated isolation from sputa and resectcd lung tissue of acid-fast bacteria.which were nonpathogenic for guinea pigs has led to a change in attitude and further attempts to evaluate the significance of these unnamed.mycobacteria. The tone "atypical” as applied to acid-fast organisms was first used by Pinner (1935). He described in detail the isolation of 15 strains from such sources as the sputum of a man with.fibrotic tuberculosis. urine samples. gastric lavage. stools. blood and from pus from a tuberculous hip joint. Colonies were colored. being lemonpyellow to dark orange. When injected into guinea pigs in small doses. no demonstrable lesions were produced. However. 10 mg. doses injected subcutaneously into guinea pigs and intravenously into rabbits and chickens gave far from uniform results. Guinea pigs injected subcutaneously developed subcutaneous nodules 3-9 dnys later which soon became abscesses. Lesions in the regional lymph node were sometimes present. The subcutaneous lesion reached its maximum size in 2-3 weeks and then receded so that it was not palpable at 5-7 weeks after injection. In some cases the abscess eroded through the abdominal well to produce a serous peritonitis. These atypi- cal.nyoobecteria.fell.into one of three classes on the basis of guinea pig inoculation studies. vis.. (a) those which elweys produced peritoneal lesions. (b) those which sometimes produced peritoneal lesions. (c) those which produced only localised lesions at the inoculation site. Wolinslq. sum. Mitchell and Steenken (1957). in reviewing the history of disease caused by the atypical myoobecteria. considered that the papers of Buhler and Pollack.(l953) and Timpe and Runyon (195k) “marked the beginning of the recognition of such cases with increasing frequency-of human disease apparently caused by acid-fast bacilli which were nonpethogenic for guinea pigs.“ I. Buhler end.Timpe (1953) described two hummn.ceses of disease from each of which was isolated an acidpfast'becillus which grew a.yellow colony. This was later identified by Runyon (1959) as a Group I myccbecterium. In the first case. the lung was resected and.was found to contain a cavity lined by chronic granulation tissue heavily'infiltrated with lymphocytes. plasma cells and occasional foreignébody giant cells. The lumen of the cavity contained necrotic debris in which there were polymorphonucleer leukocytes and monocytes. Another small nodule had an area of central caseous necrosis surrounded by epithelioid cells and giant cells. There was an outer acne of dense fibrous tissue in which there were plasma cells. The second case was that of a 21-year-old man who died. At eutcpey. excess serosenguineous fluid was present in.the abdomen. thorax and pericardium. Minute shot-like nodules were present in the lungs. The hilar lymph nodes were calcified and partly caseous. There was a caseous and liquefied lesion 1 cm. in diameter in the pancreas. The spleen was enlarged and contained numerous. well circumscribed white caseous and calcified areaa. up to 2 cm. in diameter. HistolOgicelly. the spleen end.mesenteric lymph nodes contained large caseous masses with areas of calcification and enormous numbers of acid-fast bacilli. In the lung there were sharply defined nodules of confluent areas of bronchopneumonia which consisted of mononuclear and plasma cells. Fibrosis. but no caseation. was present. Ho.tubercles or langhans' giant cells were seen in any tissue. Ialaberder (1961) spoke of the ”rediscovery in the last few’years of the unclassified so-called 'etypical' mycobecteria'. he pointed out that the attitude was to agree tacitly to regard as unclassified those acid-fast bacilli. isolated from human specimens. but whose properties are similar to those of sapro- phytic mycobecterie or closely related organisms. 5 Such a group of bacilli. pathogenic in man.but not, the guinea pig. has already been described by many workers. In a review of the literature up to the end of 1950 ... the writer has noted that 66 investigators have reported the isolation of one or more of such organisms. Runyon (1959) proposed the name “anonymous" for the unclassified nycobacterie and described a classification based on the effect of light on colonial pigment production and their rate of growth. as follows: Group I. Photochromogens: little or no pigment when grown in the dark. pigmented (bright yellow) after brief exposure to light. Later Runyon (1960) suggested that Group I photochromogens which represented a relatively homogeneous group be named 11. 553121..- Group II. Scotochromogens: yellow to pale orange when grown in the dark. more reddish if grown continuously in light. Group III. Nonphotochromogens: little or no pigment whether grown in the dark or light; if pigment present this is not as in Group I or II organisms. I Group IV. Rapid growers: little or no pigment. Runyon (1959) described in detail the relationship between the atypical mycobacteria in his collection and the patient's clinical and pathological findings. Group I nycobecteria had been recovered repeatedly from many sputa and from 3# resectcd lumps. Only 9 of the 122 patients had had infection with 11. W. The significance of Group II organisms was not clear. His collection included 26 strains recovered from cases also infected with true tubercle bacilli. There were also 96 other strains. but probably only in a very small number of patients had Group II strains detablished more than a transient residence. Of the 143 strains of Group III wcobecteria. each ms been isolated .1; least me. from different sputum samples from each patient. and most of these 6 ,. patients had serious pulmonary disease. Later. Mon (1960) said that he thought that Group I was significant in human disease but Group II was not. and that both Groups III and IV may or may not have been associated with human disease. Corps. Runyon and Lester (1963) stated that the pathological picture of disease associated with Group IV ncobacteria has not been clearly delineated but the evidence suggested that they do not prochice the characteristic features of caseating granulomatous disease. There is still not unanimity as to whether these unclassified syco- baoteria should be called I'atypics.1" or ”mow-our. Karlson (1958) challenged the use of the term 'atypical' on the grounds that they should be called W 122.. as this classification existed in the 6th edition of Bergey's Manual (Breed. Hurray a Kitchens. 19158). he 7th edition (Breed. Murray 3. Smith. 1957) makes no reference to W m. and therefore. presumably this is no longer a legitimate term. Recently. Corpe gt 1;. (1963) recommended that the term "unclassified" rather than the 'atypical" or I'anoqymous" be used. on. of the biggest drawbacks to the determination of the pathogeni- city of atypical swoobacteria is the lack of a suitable experimental animal. Mon (1959) described this thusly: The guinea pig no longer sits alone on the throne of decision as to pathogeniodty of acid-fast bacilli for man; there is the mouse. and also an empty chair. Mice have been found to be more sus- ceptible than guinoa pigs to some. of the snow-nous swcobacteria. The 'empty chair' pertains to the lack of arw known animal host. in which certain strains can establish progressive infection. al- though these bacteria evidently have been involved in human disease. Kuhica. Beam. Vestal and Pool (1960) advocated the use of intracutaneous injection of acid-fast organisms in order to determine the virulence of mycobacteria. This was based on the observations of Kite. Patnode and 7 Read (1952) and Lester (1939). Kite 2; g. (1952) injected known patho- genic mycobacteria - 11. tubergulosis (H37RV). 21- bovis (Ravenel). )3. 9.1% (Kirchberg)- and g. M in doses of 0.01 mg. in 0.1 ml. intracutaneously into the shaved abdomens of guinea pigs. In the case of the pathogens. a persistent ulcer alweys appeared in less than 10 days. Kubica gtugl. (1960) injected 0.1. 0.01 and 0.001 mg.. each in 0.1 m1. saline. into the shaved abdominal skin of a guinea pig. An ulcer at the injection site was considered to be a positive result and usually appeared in 8-1h days. and only rarely did it take 21 days. All avian. human and bovine tubercle bacilli produced ulcers. One of five saprophytes also produced an ulcer. The Groups I and III myoobacteria were the most consistent in producing ulceration. Eleven of twelve strains of Group I and seven of eleven cultures of Group III mycobacteria caused ulcers. Only two of four Group II strains and two of nine Group IV'nycobacteria gave positive results. These workers found that only those guinea pigs inoculated by the intracutaneous route with human and bovine tubercle bacilli produced progressive tuberculosis. Durr. Smith and Altman (1959) investigated the pathogenicity of some of the classical pathogenic mycobacteria. photo- chromogens and nonphotochromogens for laboratory animals. Chickens were injected subcutaneously or intravenously. Guinea pigs were injected sub- cutaneously. hamsters intraperitoneally and mice intravenously. Sapro- phytes did not produce any demonstrable disease in the animals. The photochromogens regularly caused progressive disease in hamsters and mice. Photochromogens and nonphotochronogens produced very little disease in the chicken. whereas avian tubercle bacilli produced extensive disease. Nonphotochromogens were much less virulent for any of the test animals. Pollack and Buhler (1955) described an atypical acid-fast organism which 8 they called a “yellow bacillus" and which Runyon (1959) later confirmed as a Group I nycobacterium. This organism produced progressive disease in the guinea pig. chicken ... rabbit. produced variable amounts of disease .in the mouse and rat and.consistently produced progressive fatal disease in the Syrian golden hamster. Perhaps the whole problem of animal inoculation experiments and their evaluation could not be stated more clearly than has been done by Pianos (1935): Classification as to pathogenicity is hampered by the fact that the term 'pathogenic' is nearly mean- ingless 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 demonstra- ble tissue alterations be called disease. and accordingly any organism that causes them is con- sidered 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 pgthgggnig only to those microorganisms that cause progres- sive disease. and to exclude all those that produce self-healing lesions. would exclude a major portion of all so-called pathogenic (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 criterium is used to differen- tiate between pathogenic and saprophytic acid-fast bacilli it is apparent that all non-mammalian acidp fasts belong in the saprophytic group. although it has probably never been settled whether or not some acidpfasts isolated from ooldAblooded animals are. in the sense specified. pathogenic for the respec- tive species. It may be felt that the character of the inflammatory response may . give some idea.as to the species of the nyoobacterium.involved. However. Ippen (1956) found that in domestic and laboratory animals infected with the classical pathogenic nycobaoteria. the type of organism did not alter the structure of the giant cell. but this depended on the host. 9 Feldman (1960b) has. stressed the difficulties associated with making a histologic diagnosis of tuberculosis. He states that tubercles mey be present in lymphogranuloma.inguinale. tularemia. brucellosis. tubercuioid leprosy. syphilis. typhoid fever. infection with m m. so-called 'skin tubercu- losis of cattle. sarccidosis. berylliosis. the intracutaneous tuberculin reaction. silicosis. and certain.fungous infections such as nocardiosis. The ability of the pathologist to diagnose tuberculosis was put to the test by Corps and Stergus (1963). They sent duplicate sets of 25 slides to 27 pathologists (including one veterinary.pathologist) in the 0.3.A. All the slides had been made from.surgically resectcd.human specimens. Iron some n. W had been isolated and from others the Battey strain. Group III. nonphotochromogenic sycobacteria. Only specimens from which the same strain was cultured. as was recovered from the sputum of the patient before surgery. were used. Pathologists were asked to check one of four choices on the questionnaire: (1) the histopathologic picture was compatible with tuberculosis due to n. t (2.) the histopathologic picture was compatible with tuberculosis‘dse to the Battey strain. Group III. nonphotochromqmns (3) the histopathologic picture was compatible with tuberculosis out the patholo- gist was unable to differentiate as to the causative organism (a) if the pathologist thought the histo- pathology was not due to tuberculosis he could mark it non-tuberculosis. Twentyuseven pathologists voting on 25 slides each gave a total of 675 choices. Fifty-three per cent of the votes indicated that the patholo- gist could not differentiate between.the causative organisms. Six.per cent of the votes were cast for nontuberculosis disease of which there were no cases. giving a totai.of 59"which could.not be differentiated. 0f the 29$ of the votes for g. M. 62$ of these were incorrect. In the words of the authors. 10 his study confirms the writers‘ impression that the histopathologic picture of infections caused by the Battey strain. Group III. nonphotochromo- genic swcobacteria is not distinguishable with an degree of accuracy from that caused by n. W. Corpe. Rumon and Lester (1963) in a review on the status of disease due to unclassified wcobacteria stated that The spectrum of gross and microscopic pathology produced in human tissues by infection with either Group I. II. or III wcobacteria is almost identical with thIt mane“ by no We Thus. MSW pathologic findings cannot be relied upon to differen- tiate Group I. II or III infections or to distinguish them from lesions caused by g. W. Present evidmce suggests that the Group IV qcobacteria do not produce the characteristic features of oaseating granulomatous disease. Herckx .3 g. (1963) also agreed that he was not able to tell which of the h groups of ncobacteria had caused the lesions. and in most cases these were similar to those due to n. W. Crow. King. Smith. Corps and Stergus (1957) found that the gross and histopathologic findings in 7 surgical specimens they examined were identical with those caused by B- We Perhaps the words of reldman (1960a)sunarise the present situation most lucidly: With few exceptions. the diseases produced naturally by wcobacteria can be classified properly as. infec- tious. This designation is often fortunate and convenient for the hard-pressed pathologist. who cannot alqu determine. by examination of the mor- . bid tissue. the. precise nature of the causative agent. Structural changes or patterns in the tissue that. represent the host's response to myoc- . bacteria aid to man of the fungous infections have . several cellular components in canon. The diag- nosis of such material based on histopathology alone is. slugs unwise and'often difficult. if not impos- sible. In dealing with infectious granulomas. the pathologist must recognise the diagnostic limitations of tissue microscopy. This is particularly impor- tant because the causative agent may be present in small numbers or mey be difficult to recognise unless suitable bacteriologic studies are done. 11 A national progrem.of tuberoulin.testing was established in the 0.3..A. in 1917 (Steele and.RanneI. 1958). The percentage of reactors dropped from St that.year to 0.11! in 195“ (Johnson. Baisden and Frank. 1961). The h8th state was accredited in 19h0. this meaning that less than.0.5$ of the cattle in each state were reactors to the tuberculin test (0. 3. Dept. of Agriculture. 1960). This same author states that in 1955 the number of reactors started to clinb and increased every year until in.1959. when a total of O.23$ of cattle tested were classified as reactors. Wilder (1962) points out that in 1914. $9.000 bomine car- .casses were condemned or passed for cooking only. but in 1961 only 88 were in this category. Of the 1h.000 reactors reported in 1961. less than 200 had lesions sufficiently extensive to be placed in this category. and 73$ of reactor cattle during.the 1961-62 fiscal.year did not have gross lesions of tuberculosis.(Wilder. 1962). Cattle mey be sensitised by other'nycobacteria. such as‘flt.ngg|: W. John.“ bum-o s. m n. W and «mind acid-fast qcobacteria (Johnson .3. 3.. 1961). The presence of acid- fast bacilli in.subcutaneous lymphangitis in cattle. frequently called 9skin.1esions' or "skin tuberculosis“ has been known since 1916 (Traum. 1916). These lesions hare beencdescribed.by Karlene (1962) as being . characterisedfibyuthe.ooonrrenea..in_the.sabentis.. efxfirm.or fluctuant nodules. some of which.ulcerate. The lesions usually are seen on the legs. but in. some cases the nodules appear to coincide with the lymphatic vessels along the shoulders and neck. or were rarely. the flank or back. They vary from small. almost imperceptible elevations to large masses 10 cm. or more in diameter. Hicroscopically. the lesions are typical granulomas with varying degrees of necrosis. caseation. calcification and. giant cells. .Acid-fast bacilli are seen in most skin lesions. The histopathologic picture is typical of tuberculosis. 12 A considerable. amount of work was done on this condition particularly, by Daines and.Austi.n (1932,1934) and Daines (1938). who isolated . variety of acid-fast and nonacid-fast organisms free these lesions. In reporting the results of work done in mgland. Hole and Bulse (1939) stated that. We have to admit failure of our attempts both to cultivate a causal agent of the lesions and to transmit the condition by experimental animal inoculation. The exact nature of the acid-fast organisms in "skin lesions" has yet to be determined. Other cases of the isolation of atypical acid-fast bacteria from cattle have been reported. In 1930. Hastings. Beach and Thompson reported isolating acid-fast bacteria from the lymph nodes of cattle which had reacted to the tuberculin test but which did not show evidence of tuberculosis at necropsy. Hogan (1931) also recovered acid-fast bacteria from normal mesenteric lymph nodes of cattle. These were classed as saprophytes and he postulated that as acid-fast organisms were conon in the intestine. the presence of saprophytes in the mesenteric lymph nodes was not surprising. He also conjectured that these acid-fast bacteria m be able to cause transitory sensitivity to tuberculin. In 193:0. larlson and Feldman reported recovery of nonchromogenic acid-fast microorganisms from 25$ of 91+ swine tonsils. These cultures. did not produce no recog- nisable disease in chickens. mice or calves. Smith (1951}. 1958) reported the isolation of atypical umbacteria from the lymph nodes of apparently normal cattle in kglsnd. Scammon. Pickett. Froman and Will (1963) dismissed the cultural characteristics of 1&3 cultures of acid-fast micro- organisms freshly isolated from organs of tuberculous swine obtained from a federally inspected abattoir. Most of the swine showed only caseous . lymph nodes. Hall-ans. Hallmenn and Robinson (1964) reported the isolation of 'a relatively large number of acid-fast organimas. not the classical 13 pathogens. from bovine and swine tissues.“ They selected ho cultures at random from approximately 300 isolants from animals and classified them as: 3. m. 3: Group I. 13 Group II. 1: Group III, 12: pseudochromes. 10: Group IV. ll: and saprophytes. 2. The statement of Kubica 3; g. (1960) The fact that it has been impossible consistently and repeatedly to fulfill Koch's Pbstulates as regards the anonymous and isoniasidrresistant acid-fast bacilli is no reason why we should negate their importance in human disease should be equally. applicable to the situation in the field of animal mycobacterio si s . III. MATERIALS AND METHODS was; magi Calves were obtained by Dr. R. M. Scott. Animal Disease Eradication Division. 0. S. Department of Agriculture. Lansing. from various Michigan herds which had histories of negative caudal-fold tuberculin tests. Calves were tested by the cervical method using 0.1 ml. avian tuberculin. 0.1 ml. mamalian tuberculin and 0.2 ml. Johnin. filly those that had no response to any of these three tests were selected. Calves were of various breeds - Holstein. Holstein-Angus crossbreeds. Guernsey. and Holstein-Guernsey crossbreeds - and were between 6 and 10 months old at the time of inoculation with owcobacteria. Details of the sex. breed and age at necropsy are given in the protocols. Calves were housed in an excellent isolation unit. but as it had only 10 rooms. generally several animals were placed in each room. Calves l to 6 were housed in individual rooms. but calves 7 to 58 were accommodated 3 to 5 to a room. They were kept in strict isolation until necropsied at approximately 60 days after inoculation. Their rooms were cleaned daily and by personnel who wore rubber suits. boots and disposable gloves. caps and masks. Calves were fed a balanced ration and water fl #3.. and the site of inoculation was examined closeh and other clinical observations made once per week. Approximately 50 days after injection with the organisms the calves were tuberculin-tested. ll} 15 o o s Calves were inoculated with cultures of g. 9353. L1. £132- and atypical wcobacteria. The atypicals were classified as Runyon Group III mycobacteria of bovine origin. swine origin or feed or soil origin. 'pseudochrome" Group III noobacteria or Group IV sycobacteria. The cultures. supplied by Dr. V. Mallmann. were as follows: Group III bovine origin: 510-0. 680-0. SOB-O. 62D-0. 1078-0. 710-0 and 783-0. Group III swine origin: 9.30-0 1720-.1 1730-1. 19302-1. Group III feed or soil origin. Group III pseudochrome mycobacteria: 523-1. 613-0. 11213-0. 128F-0. Group IV mycobacteria: thrF-O. lit-l. 8711-0. 1173-0. 7F-l. 2 flit-l. These atypicals were classified. on the basis of the classification of Runyon (1959). Group III mycobacteria differ from the classical pathogenic wcobacteria. 1.... n. m. a. W and g; m. in that they do not initially produce progressive disease in guinea pigs. rabbits or chickens. The colonies are nonpigmented whether they are grown in light or in the dark. The optimum temperature is 35 C. to 37C.. but theymaygrowat 22. 30andevenh5 C. Theyareniacin- negative. generally neutral red-negative. produce a catalase which is inactivated at pH 7 ande 5 in 20 or 25 minutes at a temperature-cf 60 C.. and do not form serpentine cords. They may be uniform or long beaded acid-fast rods. All those of bovine or swine origin produced an ulcer at the site of inoculation when 0.1 mg. was injected intradermally into guinea pigs. Those mycobacteria of feed or soil origin produced no . intradermal ulcer. Hycobaoteria producing an ulcer also induced delayed sensitivity. In most cases the sensitivity was greater to avian tubercu- lin than to mamalian. Two cultures of bovine origin induced a 16 sensitivity greater to PPD-B. a purified protein derivative prepared from a human Battey strain isolant. Pseudochromes The pseudochromes are similar to Group III except: (a) while no pigment is produced by young cultures. on continued incubation. particu- larly on a moist slant or in a broth. a light yellow pigment is produced (these may be identical to organisms which have been classified by others as "pigmented Group III sycobacteria' or "Group IIIb'): (b) most of them do not produce an ulcer at the site of intradermal inoculation in guinea pigs. Generally sensitivity is induced. Group IV Mycobacteria These differ from the classical pathogenic mycobacteria. Group III mycobacteria and pseudochromes in their rate of growth. requiring only 2-5 days for isolated colonies to appear on Lowenstein-Jensen (Difco) slants at 35-37 C. They differ from the saprophytic myoobacteria such as 5. M in that they generally do not grow at M C. and generally are not as strongly arylsulfatase-positive. They may or may not be pigmented and generally do not produce ulcers at the site of intra- dermal inoculation in the guinea pig using 0.1 mg. wet weight of organisms. Calves l to 6 received a total of 2.2 mg. wet weight of the organisms intradermally. me milligram was given in'tradermally proximal to the carpus on the medial aspect of the left foreleg and another 1 mg. was given intradermally just distal to the hook on the medial aspect of the right hindleg. One-tenth milligram was also injected.intra- dermally distal to the carpus on the medial aspect of the left fore- leg and a similar amount proximal to the hock on the medial aspect of l? the right hind leg. 'Except for calves #7 and #9. which received 10 mg.. wet weight. of organisms. calves 7 to 50 received 1 mg.. wet weight. of organisms intradermally on the lateral aspect of the left foreleg just proximal to the carpus. Calves 51 to 58 received a total of 10 mg.. wet weight. of organisms each. 2 mg. being given by each of the following routes: oral. intredermal at the same site as calves 7 to 50. subcu- taneous. behind the right elbow. intramuscular. into the right gluteal mass and intraperitoneal. into the right paralumbar fossa. um mm»: The technique used was based on that of Jones and Gleiser (l95h) and was designed to allow maximum examination of the cadaver under the existing facilities. All lymph nodes were removed. placed in plastic sacs with appropriate labels and brought to the laboratory'where they . were examined minutely in a bacteriological hood by slicing them trans- versely at approximately 3 mm. intervals. Prior to killing the animal 2 2.50 ml. tubes of blood were collected for serum. and for hematological examination blood was taken from the Jugular vein into a tube containing IDTA.‘ The animal was killed by exsanguination after being anesthetised with chloral hydrate given intravenously. Fifteen milliliters of 40$ chloral hydrate were injected per 100 lbs. of body weight. After anesthesia was induced. both the left and right common carotid arteries were dissected free and incised to give maximum exsanguination. The head of the animal was raised to prevent the accidental inhalation of blood. The ear tags were removed. numbers recorded. and both the tags were placed in the bottle with the corresponding specimens. The right foreleg and right hind leg were removed and the following lymph nodes dissected free. then placed in.plastic sacs: right prescapuler. right *Rthylenediaminetetraacetic acid. 18 axillary. right prefemoral. right popliteal. right ischiatic. and left and right superficial inguinal lymph nodes. The right humerus was isolated and 3 inches of it was cut from the proximal half for later removal of the bone marrow. The skin over the lateral surface of the body was reflected dorsally. The abdomen was opened from the dorsal and of the costal arch to the xiphoid process and then along the lines alba to the pubis. and the freed abdominal wall was reflected dorsally. This method allowed access to the deep inguinal lymph node. The costal attachments of the diaphragm were out and the dorsal ends of the ribs were severed with rib shears. Then the distal ends were similarly out and the right chest wall was removed in 2212' The diaphragmatic lobe of the right lung and a portion of the ventral lobe of the liver were removed aseptically for bacteriological examination. The skin was reflected dorsally from the midline incision from the symphysis mandible to the thoracic opening. The tongue was freed from the mandible. and the left and right submaxillary lymph nodes were identified and removed. The soft palate was transected. the greater cornua of the hyoid bones were disarticulated from the larynx. and the neck organs were dissected free. These and the lungs were removed in 3&9. and placed on the table. The left and right lateral retropharyugeal lymph nodes and the left and right medial retropharyugeal lymph nodes were removed from the head and neck. From the thorax the posterior mediastinal. left and right bronchial and anterior mediastinal lymph nodes. including the node at the bronchus to the right apical lobe. were removed with the minimum of contamination and placed in separate sees for bacteriological examination. The esophagus and trachea were opened throughout their lengths. and the lungs were sliced transversely at approximately 2 cm. intervals. 19 Meanwhile. the assistant had removed the small intestine. as described by Jones and Gleiser (195“). as this method left all the mesenteric lymph nodes together attached to the mesentery. These were removed in, 3232 and the lymph nodes then dissected free. The kidneys were isolated and then the forestomechs and spleen together. The latter was freed and half of it was collected for bacteriological examination. The lymph nodes of the forestomechs were removed and pooled. The liver and diaphragm were removed together by cutting through the diaphragm's attachments. The hepatic lymph nodes were dissected free. Then the left and right deep inguinal lymph nodes and internal iliac lymph nodes were removed. The head was removed and the left and right parotid lymph nodes were collected. The brain was removed. The car- cass was turned onto its right side. and the left prefemoral. left popliteal. left axillary and left—prescapuler lymph nodes were removed. The left prescapuler node and the skin at the site of inoculation were taken for bacteriological examination. The left humerus was collected in a manner similar to that used for the right. Whoa comm lymph nodes were sliced transversely at 2-3 mm. intervals. and slices from the following nodes were fixed in 10% buffered neutral formalin: left and right parotid. left and right submaxillary (mandibu- lar). left and right medial retropharyugeal (suprapharyngeal). left and right lateral retropharyugeal (atlantal). left and right prescapular. left and right axillary. anterior mediastinal. posterior mediastinal. left and right bronchial. hepatic (portal). mesenteric. colic and 'forestomach'. internal iliac. left and right deep inguinal. lumbar .chain (if affected). left and right external inguinal (superficial 20 inguinal). left and right prefemoral. left and right ischiatic and left and right popliteal lymph nodes. Also fixed in formalin were sections of liver. lung. heart (right ventricle). both kidneys. spleen. brain. ileum and skin from the site of inoculation. Identification of the lymph nodes was based on the classification of Baum (1912). The relationship between the terminology of Sisson and Grossman (1953) and Baum (1912) has been discussed by‘webb (199“). Also duplicate blocks of heart. lung. spleen. liver and.ileum were fixed in Zenker's fluid (without the addition of the acetic acid). loch brain was sliced transversely at approximately 0.5 cm. intervals by the technique described by McGavhu. Rsmby and Tammemagi (1962). and blocks were cut from the following sites: medulla oblongata at the level of the pyramidal decussation. medulla oblongata at the level of the obex. medulla oblongata in the middle of the fourth ventricle. cerebellum through the middle cerebellar peduncles. midbrain at the level of the rostral colliculi. corpus striatum at the level of the trigone olfactorium. cerebral cortex.from the frontal and occipital poles and cerebral cortex (parietal) at the same level as the corpus striatum. All tissues were embedded in paraffin. cut at 6 microns and stained with new fuchsin-hematoxylin and eosin (Hilligan. Garric and Trosko. 1961). Selected lesions were stained by a Crossman's modifi- cation of the trichrome stain (U. 3. Armed Forces Institute of Pathology. 1960) and for reticulum by the method of lesser and Shanklin (1961). In evaluating the results. the lesions have been classified in one of the following four categories: 21 (1) no gross or microscopic lesions (2) lesions at the skin inoculation site only (3) lesions in the regional lymph node draining the skin inocu- lation site with or without detected.lesions at the skin inoculation site. i.e.. a primary complex (h) generalised lesions. i.e.. lesions more extensive than the primary complex. Attempts were also made to determine whether the lesions were . progressive or not on the basis of the criteria laid down by Feldman (l9h3) for guinea pigs. He stated that the signs of progressive lesions were: (1) peripheral extension of the disease with daughter tubercle formation (2) confluence of morbid tissue (3) conglomerate tubercles (h) slight to extensive necrosis Nonprogressive lesions were characterised by: (l) peripheral encapsulation without daughter tubercles in the peripheral some of the capsule (2) presence of noncaseating tubercles (3) evidence of the transition of epithelioid cells to fibro- blasts ' (it) fibrosis (5) calcification or bone where there are no signs of progressive tuberculosis 22 WW Tissues removed at necropsy with a minimum of contamination were taken to the bacteriological laboratory in plastic sacs. Here they were trimmed free of excess adipose tissue and soaked in 5‘ sodium hypochlorite solution. 5 changes of 5 minutes each. Then they were sliced under a bacteriological hood. using aseptic technique and fresh sterile instruments for each pool of tissues. The tissues were cultured separately or in pools as follows: (1) left prescapuler lymph node (2) skin at the site of inoculation (3) anterior mediastinal and left and right bronchial lymph nodes (h) posterior mediastinal lymph nodes (5) liver and spleen (6) lung (diaphragmatic lobe of the right lung) (7) hepatic lymph nodes (if lesions) (8) bone marrow from both the left and right humeri Tissues were blended with nutrient broth in a‘waring blender and a 10 ml. amount was transferred to a sterile tube. An equal volume of #5 Neon was added to the tissue broth homogenate. After shaking. it was allowed to stand for 15 minutes. and then the pH of the solution was adjusted.to 7 with 2! BCl. After centrifugation. the supernatant ‘was removed and the sediment was seeded onto slants of each of Lowenstein- Jensen medium. Middlebrook 7H10 agar and Dubos Gleic agar. Wham: Approximately 50 days after inoculation. calves were tuberculin- tested in the caudal fold with 0.1 cc. mammalian tuberculin and in the cervical region with 0.1 cc. avian tuberculin. 0.1 cc. mmmmalian 23 tuberculin and 0.2 cc. johnin. The mammalian and avian tuberculin used ‘was 'heat concentrated synthetic medium tuberculin" made to the speci- fications of the Agricultural Research Service. 0. S. Department of Agriculture (Anon.. 1962). The skin thickness was read with calipers at each site immediately before injection and at 2“. #8 and 72 hours after injection. In the cervical region the avian site was the most anterior. the mammalian in the middle and the johnin the most posterior on the lateral surface of the neck. These techniques and their interpretations were based on the recommendations of Boddie (1962) and Anon. (1962). ammonium A blood sample was taken from.the jugular vein immediately prior to necropsy into a bottle containing IDTA as an anticoagulant. Hemo- globin concentration. hematocrit and a total leukocyte count were determined. The eyanmethemoglobin and the microhematocrit methods were used (Benjamin. 1961). The leukocyte count was done using a Neubauer hemocytometer and Turk's diluting fluid. TABLE I. 24 Index to incculum. group. calf no. and page no. in results. Chlf I—iage Calf F:E;-' go. Igggglya 9:232 Ho. 0 0 Ion iflg‘__ 1 508-0 III #9 18 7F-l IV 87 2 sic-0 III 26 20 81-0 M. m 1 100 3 620-0 III 50 21 a. my; 96 1. 680-0 III 39 22 878-0 IV 8“ 5 thF-O IV 82 23 17201-1 III 61 6 1301-0 5. m 98 21: 1730-1 III 611 7 71C-O III 83 25 ZSHF-l IV 93 9 tar-1 IV 83 26 81-0 M. m 102 10 788-0 III 59 27 g. m 97 11 528-1 Pseudochrome 76 28 1178-0 IV 85 12 878-01 IV 83 29 51C-0 III 28 13 1073-0 III 55 30 17201-1 III 62 11: 613-0 Pseudochrome 79 31 172c1-1 III 63 15 1173-0 IV 84 32 193c2-1 III 65 16 930-0 III 60 33 193c2-1 III 6.6 17 1128-0 Pseudochrome 81 34 1860-1 III 69 25 TIBLE I-Continued F 1 p... ‘F Page low- is... o 0 n- u o 35 167C1-1 III 69 #8 Soil orig. III 7# 36 1860-1 III 68 09 Soil orig. III 7!: 37 167C1-1 III 70 50 Soil orig. III 75 38 7lC-O III 57 51 7F-l IV 89 39 7lC-0 III 58 52 117B-0 IV 85 #0 1078-0 III 55 53 25uF-l IV 93 #2 510-0 III 29 5a 251ml Iv 9‘: #3 51C-0 III 31 55 618-0 Pseudochrome 80 ## 62D-0 III 52 56 128F-0 Pseudochrome 81 #5 680-0 III #0 57 528-1 Pseudcchrome 77 #6 Feed orig. III 72 58 528-1 Pseudochrome 78 #7 Feed orig. III 73 IV. RESULTS Fifty-five calves were injected with sqcobacteria. these being atypical wcobacteria of bovine origin. swine origin and feed and soil origin. as well as a. m and fl. 53.1.20 The results are subdivided according to the cultures used. " 3 Group III mcobacteria of Bovine Origin . Fifteen calves were inoculated with cultures of Group III mycobac- teria of bovine origin. as follows: Culture Number Calf Number 510-0 Calves 2. 29. #2. 43 680-0 Calves #. #5 508-0 Calf l 62D-0 Calves 3. ## 1078-0 Calves l3. #0 710-0 C8170! 7o 38: 39 788-0 Calf 10 Missal—1 -0 9g; 2 - 2.2 mg. inoculum intradermally. mu stamina:- None. mill-ble. W W. (72 days after inoculation) Holstein heifer. 8 months old. lesions were produced at both sites of inoculation in the left foreleg. The upper lesion consisted of a 1 cm. diameter encapsulated focus of yellow caseous material. The lower lesion also showed no ulceration to the surface and was well encapsulated and contained a thin yellowish- white material. he calcification was seen. There were also two 26 27 inoculation sites on the right hindleg. At the upper site. a 15 mm. diameter encapsulated lesion contained creamy yellowish-white material. The lesion at the lower inoculation site on the right hindleg consisted of a 5 -. diameter nodule. The anterior and posterior mediastinal and left and right bronchial lymph nodes contained numerous discrete and confluent yellowish-white foci. some of which were calcified. (he of the mesenteric lymph nodes also contained a discrete yellowish-white focus. W W- Skin. upper. fonleg. In the dermis there was an encapsulated granu- loma which consisted of a peripheral fibrous capsule about a layer of lymphocytes. Internal to this was a layer of macrophages which enclosed a central core of caseous material in which there was some early calci- fication. Skin. upper hind leg. An intradermal. grenuloma of epithelioid cells and numerous giant cells of the Langhans' type was 1 present. Cessation was minimal. but. where present. calcification was well advanced. Acid- fast bacteria were frequently visible in the epithelioid cells. Skin. lower hind leg. This lesion showed only a small encapsulated epithelioid-cell granuloma. without necrosis . caseation. calcification. acid-fast bacteria or giant cells. left prescapular lymph node. Nonprogressive gramlomas with central caseation. calcification and well. developed epithelioid and giant cells were seen. Numerous acid-fast organisms were present at the periphery of the caseous debris. 28 Anterior and posterior mediastinal and left and right bronchial lymph nodes. Hiltiple progressive granulomas. apparently early in development. were seen. These consisted chiefly of epithelioid cells with a few heterophils lying in the centers where necrosis was commencing. Caseation and calcification were absent. Acid-fast organisms were infrequently seen. in the centers of the granulomas. Mesenteric lymph nodes. Granulomas in varying stages of develop- ment were seen in these lymph nodes. A few heterophils were present in the centers of early lesions and older lesions showed caseation and calcification. Acid-fast organisms were rare and. when seen. lay in the necrotic material. Right popliteal lymph node. Early granulomas consisting of epi- thelioid cells with occasional heterophils were seen. lo giant cells. «nation or calcification were present. Acid-fast bacteria were rarely seen in the centers of the granule-as. mm m. Acid-fast bacteria were recovered from the pool of the head and neck lymph nodes. the pool of the anterior and posterior mediastinal and left and right bronchial lymph nodes. lung. mesenteric lymph nodes. right internal iliac. right pref-oral lymph node. right popliteal. left prefemoral lymph node and the skin inocula- tion sites. EL: 32 - 1 mg. inoculum intradermally (killed). M W. The skin inoculation site never ulcerated. Swelling reached a maxi-1m diameter of 10 n. on the 21st day after inoculation and slowly regressed so that a 5 mm. diameter nodule was all that remained at necropsy. 29 m m. (5‘ days after inoculation) Guernsey heifer. 8 months old. good condition. 350 lb. weight. The only lesion detected was the 5 u. intradermal module on the cut surface of the skin at the site of inoculation. W W. A diffusely infiltrating granulomatous inflammation was. seen in the reticular lqer of the skin at the site of inoculation. It consisted of macrophages. lymphocytes and some plasma cells and was not encapsulated. W W Acid-fast bacteria were recovered from the left prescapular lymph node. from the pool of the anterior and posterior mediastinal and the left and right bronchial lymph nodes. and from the bone marrow of the humcri. 9g 3g - 1 mg. inoculum intradsrmally. This differed tree that used in Calf l in. that it had boon stored in the refrigerator for a further 10 months. m W. A diffuse hard swelling l5 -. in diameter was present at the skin inoculation site seven days after inoculation. Ten days later this had reached a diameter of 25 In. and was still hard. but the central area showed a 10 III. diameter hairless area without ulceration. 0n the 2#th in after inoculation. the swelling was reduced to 10 am. in diameter and 5 mm. in elevation. and the hairless area was now 7 n. in diameter. w the 33rd day after inoculation. the lesion had ulcerated and the swelling now occupied a 15 mm. diameter area with an elevation of 5 ms. Granulation was taking place around the periphery of the ulcer. (hi the #9th day after inoculation the injection site was represented by a nodule 10 an. in diameter with an elevation of 5 m. 30 The ulcer had healed so that the lesion was now completely closed. the central part of it being occupied by a hairless area 3 mm. in diameter. Eggzgpgx,£ingiggg. (66 days after inoculation) Holstein heifer. 9 months old. good condition. #50 lb. weight. Gross lesions were confined to the left prescapular lymph node and to the skin at the site of inoculation. A cross section through the skin inoculation site showed a pink nodule 3 mmo in diameter lying in the dermis. The left prescapular lymph node contained a caseous lesion 20 mm. in diameter. fiistopgtholggic findings, In the skin at the site of injection. a granuloma was found lying in the reticular layer. This consisted chiefly of epithelioid cells with a few giant cells and some scattered lympho- cytes. A fibrous capsule enclosed the granuloma and no acid-fast bacteria were seen. left prescapular lymph node. Multiple. sometimes confluent foci of granulomatous inflammatory tissue were seen in the section. These were frequently encapsulated and often contained a central caseo-calcareous focus. Epithelioid cells and giant cells were numerous. and acid-fast bacilli were frequently seen lying in the cells adjacent to the caseous material. W W. Acid-fast bacteria were recovered from the anterior mediastinal and left and right bronchial lymph nodes. the pos- terior mediastinal lymph nodes. the left prescapular lymph node. the skin inoculation site. the right diaphragmatic lobe of the lung. the hepatic lymph nodes and the pool of the liver and spleen. 31 Calf fl - 1 mg. inoculum intradermally. . This organism was the isolant from the left prescapular lymph node of calf 2. W W. (h the 7th dny after inoculation the skin at the injection site had a swelling 15 u. in diameter. Ten days later“ the swelling had reached a diameter of 30 mm..was very hard. and a hairless area 10 mm. in diameter which had not ulcerated lay in its center. m the Zkth day after inoculation. the swelling had reduced to 15 In. in diameter with an elevation of 5 mm.. but had ulcerated through an orifice 10 mm. in diameter which was already granulating. he left prescapular lymph node was swollen. Nine days later the swelling and the ulcer were little changed. but the prescapular lymph node was more markedly swollen. By the 39th day after inoculation the ulcer had reached a diameter of 15 ms. in the swelling which was now 25 mm. in diameter. authe thqtheulcerhadchangedlittleandwas 15mm. indiameter and still open. The total swelling was 25 mm. in diameter with an elevation of 5 mm. and the prescapular lymph node was grossly enlarged. broughout this period the animal's temperature did not exceed 102.3 and no pyrexia was noted. m m. (68 days after inoculation) Holstein heifer. 8 months old. fair condition. 500 lb. weight. Widespread cane-calm lesions were found in the left and right bronchial lymph nodes. in the anterior and posterior mediastinal lymph nodes. in the left and right prescapular lymph nodes. hepatic lymph nodes. the mesenteric lymph nodes and in the liver. lungs and the skin at the site of inoculation. 32 Skin at the site of inoculation. The ulcer measured 10 mm. in diameter and lay in the middle of an area of swelling 20 mm. in diameter. On cross section. the swollen area of skin was seen to be deeply reddened and contained yellowish caseous areas. Left prescapular lymph node. The left node was very difficult to out. due apparently to fibrous tissue. and contained multiple large areas of yellow caseous material. Right prescapular lymph node. Several.yellow foci 2 mm. in diameter were seen in the cortex.at one pole. Anterior nediastinal lymph nodes. These consisted of four lymph nodes. two of which showed no lesions. The other two were filled with both discrete and confluent caseous foci. Posterior mediastinal lymph nodes. There were five lymph nodeq.all of which contained both discrete and confluent caseous foci. Left and right bronchial lymph nodes. These were both filled throughout with yellow caseous material. Mesenteric lymph nodes. Three yellow foci. 2 to 3 mm. in diameter were found scattered throughout the whole length of the mesenteric lymph nodes. Hepatic lymph nodes. All four hepatic lymph nodes contained numerous caseous foci varying in diameter up to h mm. Liver. The only lesion found was a white area approximately 3 mm. in diameter lying subcapsularly. Lung. numerous clear glistening foci l to 2 mm. in diameter were scattered throughout the lung. lying both in the substance and subpleurally. 33 Histgpathologic m. Besides the gross lesions. microscopic evidence of tuberculous inflamation was found in the left and right medial retropharyugeal. colic and left and right deep inguinal lymph nodes. Skin at the inoculation site. The section of skin showed numerous focal or circumscribed granulomas lying in the reticular layer. These varied greatly in sise and probably represented part of a multiloculated granuloma. The larger granulomas had caseous or casec-calcareous centers and giant cells were very numerous. The fibrous tissue about the lesions appeared to be part of the reticular layer of the skin rather than a specifically developed capsule. . Left prescapular lymph node. The lesions in this lymph node were also similar to those seen in the posterior mediastinal lymph node. the granulomas being of the two types - those with central caseation and calcification and a peripheral fibrous capsule (Figures 1 and 2) and the unencapsulated noncaseous granuloma. Heterophils were present only about the calcified centers. lo acid-fast bacteria were seen. Right prescapular lymph node. Lesions in this lymph node were similar to those seen in the posterior mediastinal lymph node. They were either focal encapsulated granulomas with caseo-calcarecus centers or. noncaseous granulomas without encapsulation. Heterophils were seen only about the central calcified material. Acid-fast bacteria were rare and. when seen. lg in giant cells. Left and right medial retropharyugeal lymph nodes. Tuberculous noncaseating microscopic granulomas were found in the cortex (Figure 3). These consisted of epithelioid cells and a few giant cells; caseation and calcification were absent. Acid-fast bacteria were rare and were seen only in a giant cell. Figure 1. Left prescapular lymph node from calf #3, inocu- lated with 510-0. a Group III mycobacterium. A caseo- calcareous granuloma has a thin fibrous capsule (arrow) developing around it. New Fuchsia - H & 8. x50. Figure 2. Left prescapular lymph node from calf #3. inocu- lated with 510-0. a Group III mycobacterium. Note the calcified center (1). adjacent caseous material (2). epithelioid cells (3). Langhans' giant cells (u) and the thin capsule (5). New Fuchsin - H & 3. x18?. 35 Figure 3. Medial retropharyugeal lymph node from calf M3. inoculated with SIC-0. a Group III mycobacterium. Host of this field is occupied by a noncaseous granuloma consisting of epithelioid cells. New Fuchsin - H d 8. x187. Figure 5. Mesenteric lymph node from calf b3. inoculated with.Slc-0. a Group III mycobacterium. An encapsulated caseo-calcarecus granuloma lies under the lymph node cap- sule adjacent to a small noncaseous granuloma (arrow . New Fuchsin - H d 8. x50. 36 Anterior mediastinal lymph node. There was extensive involvement of this lymph node by tuberculoid granulomas. which were present in both the cortex and the medulla. lesions were focal but often confluent. hithelioid cells. giant cells. caseation and calcification were common. lIo acid-fast bacteria were seen. Posterior mediastinal lymph node. lesions in this lymph node were of two types - either the. encapsulated focal caseous granuloma or the diffuse noncaseous granuloma. Acid-fast bacteria were very rare. and. when located. were seen in giant cells. left and right bronchial lymph nodes. The lesions in these nodes were similar also to those seen in the posterior mediastinal lymph node. Acid-fast bacilli. however. were not seen. Hepatic lymph nodes. Again. the two types of granuloma as seen in the posterior mediastinal lymph node were visible. No acid-fast bacteria were detected. lbsenteric lymph nodes. Two types of granuloma. the encapsulated focal caseous granuloma (Figure 15) and the noncaseous granuloma. as seen in the posterior mediastinal lymph node were detected . Acid- fast bacteria were frequent and were located in giant cells. The lesions in this lymph node appeared to be progressive. since daughter tubercles had formed about the periphery of some of the larger tubercles. Colic lymph nodes. The lesions in the colic lymph nodes were simi- lar to those seen in the mesenteric lymph nodes - the two types of granul-a being present but the caseous granulomas far outnumbered the . noncaseous variety. Acid-fast bacteria were rare. being seen only in giant cells. left and right deep inguinal lymph nodes. Again the two types of granulomas were seen. i.e.. the noncaseous form with daughter tubercles. 37 and the encapsulated granuloma with caseo-calcareous centers. Hetero- phils were adjacent to the central deposit of calcium. Ho acid-fast bacteria were seen. Lung. The lung contained numerous microscopic noncaseous tubercles. These were frequently surrounded by lynhatic tissue. but were not encap- sulated. Acid-fast bacteria aml giant cells were relatively infrequent. These noncaseous granulomas usually occurred in the perihronchial lymphatic tissue. and in one case the tubercle had ulcerated through a bronchial wall to allow epithelioid cells and debris to fall into the lumen. Liver. There was an apparent activation of the Kupffer cells. as these were slightly swollen and appeared to be increased in numbers. There was also an increase in the number of lymphocytes in the portal tracts. Brain and meninges. The leptomeninges and some of the perivascular spaces in the brain adjacent to the pia mater were infiltrated with macrophages and lymphocytes (figures 5 and 6). These changes were seen in the medulla oblongata. cerebellum. midbrain. corpus striatum and cortex. However. the lesions were most marked in the midbrain. in the molecular layer of the cerebellum and in the leptomeninges over the medulla at. the pyramidal decussation. Some of these cuffs were three cells thick. but acid-fast bacilli were never seen. W m. Acid-fast bacteria were recovered from the left prescapular lymph node. the skin inoculation site. the pool of the anterior mediastinal and left and right bronchial lymph nodes. the pos- terior mediastinal lymph node. the hepatic lymph node. the pool of the liver and spleen and from the right diaphragmatic lobe of the lung. Figure 5. leptomeninges adjacent to corpus striatum from calf 1&3. inoculated with 510-0. a Group III wccbacterium. Note the well developed noncaseous granuloma distending the pia-arachnoid. New Fuchsia - H a 8. :50. Figure 6. leptomeninges adjacent to corpus striatum from calf 1&3. inoculated with 510-0. a Group III mycobacterium. The pia-arachnoid is heavily infiltrated by epithelioid cells and lymphocytes. New Fuchsin - H a I. 1:187. 39 Mm éékg _C_a_._1_f_§_- 2.2 mg. inoculum intredermally. m W. None available. uggzgpgy,fip§ingg. (82 deys after inoculation) Holstein heifer. 7 months old. The only lesions. detected on necropsy were those at the left upper foreleg inoculation sites and in two of the lymph nodes of the pool of the anterior and posterior mediastinal and left and right bron- chial lymph nodes. The skin lesion on the left foreleg of the upper inoculation site showed a well encapsulated focus 10 mm. in diameter which contained brownish-yellow caseous material. The lower inoculation site on the left foreleg was similar to that above hit was only 5 II. in diameter. Two of the lymph nodes of the anterior and posterior medi- astinal and left and right bronchial lymph node pool contained yellow caseo-calcareous foci 2 me. in diameter. gistgpathglong m. The lesions in the thoracic lymph nodes were multiple granulomas .of two types: (a) small noncaseous granulomas consisting of epithelioid and giant cells. and (b) encapsulated granulomas with central caseation. calcification and peripheral fibrous encapsula- tion. Acid-fast bacteria were rarely seen in the encapsulated lesions. but were more frequent in the noncaseous granulomas. ,Histological preparations were made only from the upper and lower inoculation sites of the foreleg. At the lower inoculation site there was a noncaseous granuloma lying in the reticular leyer of the skin. This consisted of epithelioid cells and lymphocytes. and acid-fast bacteria were readily visible. particularly in the epithelioid cells. The upper inoculation site had a granuloma with central caseation. but without any peripheral no encapsulation lying in the reticular layer. Acid-fast bacteria were plentiful. particularly in the necrotic center. but were also found scattered throughout the granuloma. W findings. Acid-fast bacteria were recovered from the left Prescapular lymph node. the right popliteal lymph node. the pool of the anterior and posterior mediastinal and left and right bronchial lymph nodes. the right internal iliac node. the left popliteal node and the skin inoculation sites. both upper and lower. on the foreleg. ga_l_f_ 131 - 1 mg. inoculum intradermally. This organism was the isolant of culture 680-0 from the left prescapular lymph node of calf h. M obsmgtion . 0n the seventh day after inoculation the swelling at the inoculation site had reached a diameter of 50 mm. and was hard. The left prescapular lymph node was enlarged. Ten days later the swelling had reached a diameter of 30 mm. and was still hard and somewhat diffuse. but in its center was a hairless area 10 n. in diameter which as yet had not ulcerated. m the 215th day after inocula- tion. the swelling now covered an oblong area 50 me. by 30 -.. with an elevation of 10 mm. The central hairless area still measured 10 mm. in diameter and was still closed. The left prescapular lymph node was swollen. 0n the 32nd day the lesion had ulcerated to the surface through an orifice 10 mm. in diameter and the swelling now measured 30 mm. by 25 -.. with a 10 an. elevation. Q: the 39th day after inoculation there was little change. but on the l$9th day the swelling was 30 m. in diameter with an elevation of 10 mm. . and the ulcer. which was still 10 mm. in diameter. was red and granulating. The left prescapular lymph node was still swollen. 41 Hgg;gpgy,fingipgs. (65 days after inoculation) Holstein heifer. 8 months old. fair condition. 350 lb. weight. Lesions were found in the left axillary. mesenteric. hepatic. anterior’mediastinal. posterior mediastinal. left and right bronchial and left prescapular lymph nodes. in the lungs and at the site of inoculation in the skin of the left foreleg. Skin. at the inoculation site. A granulating ulcer 10 mm. in diameter was found lying in the middle of the area of thickened skin which measured 35 mm. in diameter. 0n cross section this skin was found to contain patchy caseo-caloareous material similar to that seen in the left prescapular lymph node. Left prescapular lymph node. This measured 110 x 65 x 55 mm. and was affected throughout with multiple caseoécalcareous foci many of which had become. confluent (Figure 7). Left axillary lymph node. The cut surface showed numerous yellowish foci 0.5 to 1 mm. in diameter throughout the cortex. Anterior mediastinal lymph nodes. Of all the 6 lymph nodes. 5 showed no gross lesions. The largest node was filled with multiple yellow caseo-calcareous foci. Posterior mediastinal lymph node. All h nodes were filled with numerous discrete or confluent. yellow caseo-calcareous foci. Left and right bronchial lymph nodes. Both these nodes contained numerous discrete or confluent caseo-calcareous foci. Hepatic lymph node. Scattered 1 to h mm. in diameter caseous feel were seen throughout all these nodes. Hesenteric lymph nodes. In the whole chain of mesenteric lymph nodes only 2 foci were found. Each of these had originally consisted of several yellowish-white foci 0.5 to 1 mm. in diameter which had now l‘l!hl'l'||'.|'|"l.“.' c (a (4 Figure 7. Left prescapular lymph node from caIY*ES. inoculated with 680-0. a Group III mycobacterium. Note the obliteration of the lymphoid tissue by granulomatous tissue. 'I"NINIIIIlnplnlllnll'fl’q' allgullglllllllllpll - * .4 5' 6-,.7 ./' 3» Lung from calf #5. inoculated withi680-0; h Note the pale subpleural Figure 8. Group III mycobacterium. granulomas. “3 become confluent so that each now occupied an area approximately 5 mm. in diameter subcapsularly. Spleen. Several yellow foci approximately h mm. in diameter were found.throughout the parenchyma. Liver. Yellow foci b mm. in diameter. as seen in the spleen. were scattered throughout the liver parenchyma. Lung. Numerous grey foci 0 mm. in diameter were seen scattered throughout the parenchyma and subpleurally in the lungs (Figure 8). These were uniform in distribution. although they were slightly more concentrated in the apical lobes of both lungs. Surrounding each of these grey foci. which often resembled bullae. was a rim of dark red lung tissue which was presumably atelectatic. flistcpgthologic‘gingingg. Lesions were present in the colic‘lymph nodes as well as in those listed above under necropsy findings. In all lymph nodes. multiple foci of tuberculous granulomatous inflammatory tissue were seen. Giant cells were extremely numerous and caseation and calcification were present. Despite the extensive pathological changes. acid-fast bacteria were not seen in any lymph node. Left prescapular lymph node. Both discrete and confluent caseo- calcareous granulomas (Figures 9 and 10) were present. Giant cells and epithelioid cells were numerous (Figure 10). but no acid-fast bacilli were detected. Noncaseous granulomas were present in the thickened capsule of the lymph node (Figure 11). Left axillary lymph node. The microscopic lesions here were similar to those seen in the left prescapular lymph node. No acid-fast bacteria were detected. + _iw_ in ,,_,v , .i.a_i,,4, ,i i, ____H Figure 9. left prescapular lymph node from calf #5. inoculated with 680-0. a Group III mycobaoterium. Two confluent caseo-calcareeus granulomas. New Fuchsin - H a 3. x75. Figure 10. Left prescapular lymph node from calf #5. inoculated with 680-0. a Group III mycobacterium. A higher magnification of portion of Figure 9. to show the central calcified area (1). the surrounding caseous . material (2). and the granulomatous tissue (3). New Fuchsin - H l I. x187. 1+5 Left and right bronchial lymph nodes. Progressive tuberculous granulomas consisting of focal and confluent areas were widespread throughout the section. Giant cells were numerous and caseation and calcification.were extensive. No acid-fast bacteria were seen. Anterior and posterior'mediastinal lymph nodes. Again the lesions were similar to those seen in the bronchial.lymph.nodes. In none was there any attempt at encapsulation (Figures 12. 13 and lu). Daughter tubercles were readily visible in the sections of the posterior mediasti- nal lymph node. Left and right medial retropharyugeal lymph nodes. Lesions in these lymph nodes were confined to two focal noncaseous granulomas. Giant cells were seen. but caseation. calcification and acid-fast bacteria were absent. Hesenteric lymph.nodes. Progressive tuberculous granulomas were seen in these lymph nodes. Many granulomas had enlarged until they had coalesced with adjacent ones. There was no attempt at fibrous encapsu-‘ lation. and daughter tubercles were frequent. flaw of the granulomas showed caseation and calcification and heterophils were often associated with the central area of calcification. No acid-fast bacteria were seen. The colic lymph nodes. Two focal caseo-calcareous granulomas were seen in these lymph nodes. These were of microscopic size and.only a few early attempts to form small giant cells were visible. No acid-fast bacteria were detected. Liver. Tubercles consisting.of both noncaseous and caseous granu- lomas were seen in the liver. The noncaseous granulomas contained giant cells and epithelioid cells and were surrounded by a peripheral rim of lymphocytes. The caseous granulomas were enclosed by fibrous capsules and contained epithelioid and giant cells. caseation and calcification (Figure 15). Figure 11. left prescapular lymph node from calf #5. inoculated with 680-0. a Group III myoobactorium. Two noncaseous granulomas (1) lie in the thickened capsule of the lymph node (2). Under this a thin rim of granulomatcus tissue (3) surrounds the caseous area (#). New Fuchsin - H&l.x50. v— , 777 — _ _, Figure 12. Posterior mediastinal lymph node from calf #5. inoculated with 680-0. a Group III myoobacterium. A casoo-calcareous granuloma (l) and a caseous granuloma (2) lie close to one another. New Fuchsin - H & E. 875. Figure 13. Posterior mediastinal lymph node from calf #5. inoculated with 680-0. a Group III mycobacterium. Note the central calcified area (1) surrounded by a small rim of caseous material (2). which is encompassed by the granu- lomatous tissue (3). There is no evidence of the formation of a fibrous capsule. New Fuchsin - H.‘ E. x187. i S .,4 ‘0 Figure l#. Posterior mediastinal lymph node from calf #5. inoculated with 680-0. a Group III mycobacterium. Portion of the same field as Figure 13. enlarged to show the Langhans' giant cell. epithelioid cells and lymphocytes. N." mehsm " H & 3- x750o Figure 15. Liver from calf #5. inoculated with 680-0. a Group III mycobaoterium. A fibrous capsule (l) is being laid down around the granuloma (2). separating it from the liver parenchyma (3). Crossman's Trichrome. x75. Figure 16. lung from calf #5. inoculated with 680-0. a Group III mycobacterium. Note the well developed non- caseous granuloma. H & E - reticulum. x75. #9 lung. Seven sections of lung were examined. These showed multiple tuberculous.granulomas which were frequently adjacent to the bronchioles (Figure 16). In one case the granuloma had eroded through the bronchiole ‘wall to empty its contents into the lumen. These granulomas were lobular in distribution and frequently showed central caseation and calcification and there was some attempt at a peripheral fibrosis. Daughter tubercles had been formed by some granulomas. Acid-fast bacteria were not detected. Some subpleural tuberculous granulomas exhibited central calcification and caseation and had produced daughter tubercles which had penetrated through the pleura. Bacteriologic finding . Acid-fast bacteria were recovered from the pool of the anterior mediastinal and left and right bronchial lymph nodes. from the posterior mediastinal lymph node. the hepatic lymph nodes. the pool of the liver and spleen. from the bone marrow of both humeri. from the left prescapular lymph node. the skin inoculation site and the right diaphragmatic lobe of the lung. comm .92i§.$ - 2.2 mg. inoculum intradormally. 'Qlinigglmghgggzltigng, None available. Eggggpgy;figgiggg. (59 days after inoculation) Guernsey heifer. 7 months old. Numerous yellow caseous foci without calcification were present throughout the anterior and.postorior' mediastinal and left and right bronchial lymph nodes. No other lesions were detected. 50 Hgtopgthologig M. Numerous encapsulated granulomas with central caseous areas throughout which there was fine calcification were seen in the anterior and posterior mediastinal and left and right bronchial lymph nodes. Acid-fast bacilli were seen at the periphery of the caseous material. No other lesions were detected. W m. Acid-fast organisms were recovered from the right prefemoral lymph node. the right popliteal. left internal iliac. the pool of the anterior and posterior mediastinal and loft and right bronchial lymph nodes and the left prescapular lymph node. cum 932:9 m 3 - 2.2 mg. inoculum intradexmally. W W. I-ediately prior to necropsy the animal was depressed and was neither eating nor drinking. It was in very poor condition and. could rise only with difficulty. m M. (55 days after inoculation) Guernsey heifer. 7 months old. poor condition. The right and left apical and cardiac lobes of the lung showed a patchy rod and gray hepatisation. An abscess 20 mm. in diameter. which contained green pus. was found in the left apical lobe. In the kidneys. grey foci l to 10 u. in diameter were found throughout. lesions in the lymph nodes were confined to the right. popliteal. right internal iliac and left prescapular. These lymph nodes contained 'abscesses' which were filled with yellowish "pus". The four skin inoculation sites were still visible. Three of these consisted of encapsulated intradermal ”abscesses“. The upper one on the loft fore- limb was 25 x 35 ms. and the lower one 15 x 25 an. The lower lesion 51 on the right hind leg was 30 mm. in diameter and the upper inoculation. site consisted of a hard nodule only 5 mm. in diameter. WW- left prescapular lymph node. This contained an encapsulated granuloma which had a peripheral fibrous capsule. surrounding a lqyer of lymphocytes. Inside this was a leyor of macrophages which surrounded the central necrotic mass. No well developed sheets of epithelioid cells were seen. Few scattered Langhans' giant cells were present. Acid-fast bacteria were seen in the central necrotic mass and the macrophages bordering it. Right popliteal lymph node. The medulla of the lymph node had been obliterated by the encapsulated lesion which consisted of a fibrous wall inside of which was a wall of macrophages arranged as a palisado. No sheets of epithelioid cells were seen. Scattered fine granules of calcium were present in the capsule wall. Acid-fast bacilli were present in those macrophages adjacent to the necrotic debris. Right internal iliac lymph node. The histological changes were similar to those seen in the right popliteal lymph node. Acid-fast bacteria were seen in the periphery of the necrotic debris. Skin. lower foreleg. upper foreleg. lower hind leg. In each case. a focal granuloma consisting chiefly of epithelioid cells with some giant cells surrounding a central caseous mass. was present in the reticular 1eyer. Acid-fast bacteria were seen in the macrophages lining the central cavity. In the skin of the hind leg. at the upper inocula- tion site. a focal collection of lymphocytes and macrophages was present in the papillary leyor. Neither giant cells.caseation nor acid-fast bacteria were detected. 52 3932;101ch M. Acid-fast bacteria were recovered from the lung. left prescapular lymph node. right popliteal lymph node. the pool of the anterior and posterior mediastinal and left and right bronchial lymph nodes. the right internal iliac lymph node. the left popliteal lymph node. the upper foreleg skin lesion and the upper hind leg skin ' lesion. g y; - 1 mg. inoculum intradormally. This organism was the isolant from the left prescapular lymph node of calf 3. W W. m the semth dq after inoculation. the skin at the injection site showed a diffuse hard swelling 50 am. in diameter with an elevation of 10 mm. The left prescapular lymph node was swollen. Ten days later the swelling had reduced in diameter to 30 -.. with an elevation of 10 -.. but a central area. 15 mm. in diameter. was hairless. 0n the 23rd day after inoculation. the animal had lost condition and its temperature was 106 F. The swelling at the inoculation site was 10 mm. in diameter with an elevation of 5 am. and had ulcerated through an orifice 10 mm. in diameter. The left prescapu- lar lymph node was markedly swollen. 01 the 33rd day the lesion was little changed. but by the 39th day the ulcer was granulating and had reduced in diameter to 8 u. The prescapular lymph node was still markedly swollen. Ten days later there was no change. W W. (67 days after inoculation) Holstein heifer. 8 months old. fair condition. #00 lb. weight. no gross examination. lesions were found in the left pro- scapuler lymph node. in the skin at the site. of inoculation. and; in the left and right bronchial lymph nodes and the anterior and posterior mediastinal lymph nodes. 53 left prescapular lymph node. This measured 100 x 50 x #0 mm. and cut with difficulty. At one pole there was a yellow caseous focus measuring 20 mm. in diameter. The other caseous foci measuring 55 x 35 x 25 -. were found. one in the center of the node and one at the other pole. Anterior mediastinal and left and right bronchial lymph nodes. All 8 lymph nodes contained scattered yellow foci similar to those seen in the left prescapular lymph node. Skin. The surface of the ulcer was covered by a scab measuring 10 x 5 n. The swollen aroasbout the scab measured 15 mm. in diameter and on cross section the skin was found to contain a yellow focus 3 mm. in diameter. W W- left prescapular lymph node. The granulomas varied from small discrete microscopic to large confluent masses of granuluatous tissue. New of the smaller granulomas were either partially or com- pletely encapsulated. The large granulomas showed extensive caseation and some degree of calcification. Giant cells were frequent. Acid- fast bacteria. which were seen rarely. lay in the cytoplasm of the lenghans' giant cells. Anterior mediastinal lymph node. ally very sull granulomas were soon. and these were either noncaseous or encapsulated and caseo- A calcareous. Giant cells were frequent but acid-fast bacteria were not soon., Posterior mediastinal lymph node. lesions. in the posterior mediasti- nal lymph node were similar to those seen in the anterior mediastiul lymph node. numerous microscopic granulomas being scattered throughout 54 the parenchyma. Host of these were of the encapsulated caseo-calcaroous type. but a few noncaseous granulomas consisting of epithelioid cells with a few giant cells were also soon. The central calcification of the. caseous granulomas was quite marked. Acid-fast bacteria were not detected. left and riglt bronchial lymph nodes. The changes in those lymph nodes were similar to that seen in the anterior mediastinal lymph node. Scattered throughout the parenchyma of the lymph node were mmorous granulomas l to 2 ms. in diameter which were encapsulated. and in their centers were calcified areas which apparently had been previously caseatod. Giant cells were frequent but no acid-fast bacteria were soon in either of these lymph nodes- Skin inoculation site. Scattered throughout the reticular layer oftheskinwerenumorousgranul-aswhichvariedinsiseuptome. in diameter. These may have bom: discrete granulomas or they may have been part of a multiloculatod granuloma. New of them were noncaseous. consisting of epithelioid and giant cells. The larger ones frequently had caseous centers in which some calcification had taken place. Giant cells were exceedingly numerous. but acid-fast bacteria were .not detected. There was little change in the overlying epidermis. Liver. Scattered microscopic foci of mononuclear cells were seen throughout the lobulos and the portal tracts. W m. Acid-fast bacteria were recovered from the left prescapular lymph node. the skin inoculation site. the pool of the anterior mediastinal and the left mid right bronchial lymph nodes. from the posterior mediastinal lymph node. the pool of the liver and spleen and from the pool of the bone marrow from the left and right humori. 55 cum ABM g1}- lmg. inomlum intradormally. M W. The skin lesion at the inoculation site did .notulcerate. The swellingreachedamaxinm'di-oteroflOm onthe 7th.day after inoculation. maintained this siso until the 23rd do and than regressed so that at necropsy the inoculation site was represented by a module 5 n. in diameter. m m. (77 due after inoculation) Holstein heifer. 9 months old. good condition. #00 lb. weight. The only lesions detected were a red module 6 n. in diameter . mm. on the cut. surface of the skin at the inoculation site and a few fibrous adhesions between the parietal and visceral pleurao. W Mn- A moms comb-n was found 1m: in the reticular layer of the skin at the inoculation site. There was no necrosis. and no att-pt at encapsulation. Acid-fast bacteria were . readily visible scattered throughout the granuloma. W m. Acid-fast bacteria were recovered from the. loft prescapular lymph node and from the skin at the inoculation site. Calf go - 1 mg. inoculum. intradermally. This organism was the isolant from the left prescapular lymph node of calf 13. M W. m the eighth day after inoculation. the swelling at the skin inoculation site reached a diameter of 10 n. and was soft and fluctuating. A week later the. swelling was) still 1.0 n. in diameter but centrally there was a hairless area 3 -. in diameter. 0n the 22nd and 29th days after inoculation the skin lesion was unchanged 56 from that soon on the 15th day and the lesion was still closed. By the _ 38th day after inoculation the skin inoculation site was represented by an intradormal nodule 5 u. in diameter. Ulceration did not occur. m m. (70 days after inoculation) Holstein-Angus bull. 10 months old. good condition. 500 lb. weight. No gross lesions were detected except for a hairless area 15 mm. in diameter at the site of inoculation. Ch cross section of this .area the skin was not soon to be thickened and no abnormality could be W2 flashes:- 1'0 aim-conic ohms-s were soon. The skin section taken through the hairless area at the site of inoculation showed no abnormality. W m. Acid-fast bacteria were recovered only from the left prescapular lymph node. mm 2&2 £1 2 - 1 mg. inoculum intradormally. NW. The inoculation site had swollen to 20 n. in diameter on the seventh day following inoculation. Seven days later the swelling was 25 ... 1. diameter and had ulcerated. an the 2m. w it was 20 I. in diameter and a scab had formed over the surface. The lesion slowly regressed in siso so that. on the 58th day. a hairless area 5 u. in diameter remained. m m. (71 days- after inoculation) Holstein steer. 9 months old. good condition. 500 lb. weiyat. The only lesions detected wore a nodule 5 ms. in diameter in the dermis at the site of inoculation and two abscess-like lesions. 57 25 and 35 mm. in diameter. respectively. in the cortex of the left prescapular lymph node. Those "abscesses" were filled with a yellow flocculent 'pus'. W m. Microscopic examination showed that the two abscess-like structures seen grossly were encapsulated granulomas in which there was central necrosis. This central necrotic area was surrounded by a wall of macrophages and epithelioid cells throughout which lymphoid cells were scattered (Figure 17). Acid-fast bacteria were very rare and were located either at the periphery of tho caseous debris or in those macrophages bordering this debris. Skin inoculation site. A granuloma with an area of central casea- tion and calcification surrounded by epithelioid cells and giant cells was lying in the reticular layer. No acid-fast bacteria were seen. W9, W. Acid-fast bacteria were recovered from the pool of the anterior and posterior mediastinal and loft and right bronchial lymph nodes. from the loft prescapular lymph node and from the skin inoculation site. .MJQ- 1mg. inoculumintradermally. This inoculumdiffered from that used in calf 7 in that it had been stored in the refrigerator. W W. light days after inoculation. the swelling at the inoculation site had reached a diameter of #0 n. and was 20 m. thick. Sovmx days later there was an ulcer 10 mm. in diameter in the center of the swelling. m the 22nd day the swelling had reduced to 15 -. indiametor andwas 3-. thick. on. ulcer had closed-so that the center of the swelling was now occupied by a hairless area 6 am. in diameter“ 0n the 29th day after inoculation the swelling was 10 mm. in 58 dinoter and raised only 1 ... but the hairless area was 7 n. in diameter. By the, 38th day the swelling had completely resolved and the lesion was represented by a hairless area 10 am. in diameter. From the 8th to the 15th day after inoculation the animal had a temperature greater than 103 F.. the maximum being 103.8 on the 8th and l#th days. m m. (63 days after inoculation) Holstein steer. 10 months old. good condition. 500 lb. weight. No gross lesions were detected. A cross section of skin at the site of inoculation failed to reveal an macroscopic changes. W W. No microscopic changes were dotectodin the section .of skin taken from the inoculation site or in any other organ or lymph node. W gm. Acid-fast bacteria were recovered from the left prescapular lymph node. from the skin at the site of inoculation. from the pool of the anterior mediastinal and loft and right bronchial lymph nodes. from the posterior mediastinal lymph node. from the lung and from the pool of the liver and spleen. 3g: 32 - 1 mg. inoculum intredermally. This organism was the roisolant of 710-0 from the pool of the bronchial and mediastinal lymph nodes of calf 7. M W. Right days after inoculation. the injection site showed an actively inflamed area 20 ms. in diameter. Seven days laterthia hadreachedadiametorof 20ml. andwas 7mm. thick. In the center was a hairless area 3 mm. in diameter. By the 22nd day after inoculation. the swelling had reduced the 10 ms. in diameter and 3 u. 59 elevation. The central hairless. area was. now 5 ms. in diameter and the lesion was still closed. a: the 29th and 38th days after inocula- tion the lesions still appeared the same as on the 22nd day. W W (67 days after inoculation) Holstein heifer. 10 months old. good condition. 500 lb. weight. The only lesion detected was in the skin at the inoculation site. On cross section the skin showed a 7 x 5 -. reddeend area in the dermis. .W umuu. lam-contain the arm-1m at th- incculation site was composed chiefly of epithelioid cells as well as some giant cells. Cessation and calcification were absent. but there was some attempt at peripheral encapsulation. Acid-fast bacteria were very nmerous throughout the granuloma and were most concentrated in the Langhans' giant cells and the epithelioid cells. W m. Acid-fast bacteria were recovered fru the a loft prescapular lymph node. the skin inoculation site. the pool of the anterior mediastinal and left and right. bronchial lymph nodes and from the posterior mediastinal lymph node. Mn 283:2 m lg - 1 mg. inoculum intradexmally. WW. At-no stage did the skin inoculation site ulcerateto the surface. Theswollingreachedamaximumdiamotercf . 20 u. on the 7th day after inoculation. and thenregressed so that at necropsyanoduleonly5-.indiameterromainod. 60 m m. (73 days after inoculation). Holstein steer. 9 months old. good condition. 350 lb. weight. m cross section of the skin inoculation site. a nodule 10 x 5 mm. in diameter was seen in the 4...... Fibrous adhesions. between the visceral and parietal pleurae over the diaphragnatic lobes were present. Several encapsulated abscesses containing yellow-green pus anivaryingindiameterfromlOn. to20n. layintheleftandright diaphrapatic lobes of the lungs. W3 We Hicroscopic examination of the skin at the inoculation site showed a large focus of lymphocytes but no granu- lomawas seen. W. m. Acid-fast bacteria were recovered from the left prescapular lymph node and the skin inoculation site. Group III woobacteria of Swine Origin Eleven calves were inoculated with six cultures of Group III mycobacteria of swine origin. as follows: Culture. Number Calf Number 93C-0 Calf 16 172c1-1 Calves 23. 30. 31 1730-1 Calf 2“ 1866-1 Calves 3h. 36 16761-1 Calves 35. 37 mm 229:2 93; g - 1 mg. inoculum intradermally. M W. The skin at the injection site on the left foreleg never ulcerated. Swelling here reached a maximum diameter of 27 mm. on the 23rd door after inoculation. It then slowly subsided so 61 that at necropsy the nodule was 10 an. in diaweter. m m. (86 days after inoculation) Holstein steer. 8 months old. good condition. #50 lb. weight. The only lesions detected were in the left prescapular lymph node and at the skin inoculation site. An encapsulated 'abscess' 35 x 15 x 10 mm. was found in the dermis and on section was seen to be filled with yellow. creamy "pus“. a pink focus 3 -. in diameter was seen in the cortex of the left prescapular lymph node. W m. ‘lhe skin inoculation site showed an inflanmatory change similar to that seen in the left prescapular lynph node. This was a granulomatous. response in which there was central caseation with a little calcification. surrounded by epithelioid cells in which there were a few acid-fast bacteria. The left prescapular lymph node contained two granulomas. These showed central caseation and calcification. but giant cells were not common. Acid-fast bacteria were rarely seen: they were located in the epithelioid cells. mm m. Acid-fast bacteria were recovered from the left prescapular lymph node. the skin inoculation site and the pool of anterior and posterior nediastinal and left and right bronchial lymph nodes. 99:23:: 2391:} 9&2; - 1 mg. inoculum intradermally. . M W. The skin inoculation site never ulcerated. There was only a slight inflanatory swelling which was 2-3 m. in diameter on the 17th day after inoculation. The lesion regressed and could not be detected on the thh day after inoculation. 62 £23221! We (70 days after inoculation) Holstein bull. 6 months old. good condition. 350 lb. weight. The only lesion detected was in the lung. the right apical lobe of which was coepletely consolidated. Ho lesions were found at the skin inoculation site or in the left prescapular lymph node. W m. The only lesions detected were in the lung. A purulent bronchOpneumonia with subacute bronchitis. purulent exudate in the alveoli and marked twperplasia of the perihronchial lymphoid tissue was seen. W m. Acid-fast bacteria were not recovered. m g - 1 mg. inoculum intradernally. M W. Seven days after inoculation the skin inoculation site showed a circumscribed intradermal swelling 25 u. in diameter. A week later the swelling had not changed in site. but a red hairless area 3 II. in diameter was now visible in its center. 0n the 21st day after inoculation. an ulcer 5 n. in diameter was seen in the middle of the swelling. Twenty-eight days after inoculation the injection site showed little swelling. being only 10 u. in diameter and with only a slight intradernal thickening: but an ulcer 5 n. in diameter was still visible. as the 35th day. swelling was absent and only a hairless area 6 mm. in diameter now lay in the center of the injection site. This hairless area had reduced to 5 mm. on the Mind day and was still visible when the animal came to necropsy 56 due after inoculation. 63 Egg-gm finding . (56 days after inoculation) Holstein-Hereford steer. 8 months old. good condition. ltOO lb. weight. The only lesion seen was the hairless area at the site of inoculation into the skin of the left foreleg. Ch incision. a red nodule 3 mm. in diameter was visible in the dermis. W W. The only changes found were at the skin inoculation site. A granuloma here exhibited epithelioid cells. lympho- cytes. numerous giant cells and a little caseation and calcification. Acid-fast bacteria were not seen and there was no attempt at fibrous encapsulation. W m. Acid-fast bacteria were isolated from the left prescapular lymph node. the skin inoculation site. the pool of the anterior and posterior mediastinal and left and right bronchial lymph nodes. and a pool of liver and spleen. 9‘1; 3; - 1 mg. inoculum intradermally. M W. The changes at the skin inoculation site were very similar to those of calf 30. which was also inoculated with the same organism. the skin lesion ulcerating on the 21st day after inoculation. (in the 7th day after inoculation the swelling had reached a diameter of 20 ms. Seven days later it had reached a diameter of 25 -.. and in the center was a red hairless area 7 mm. in diameter. Unlike calf 30. after the lesion. had ulcerated to the surface the swelling did not subside. It reached a sise of 1+0 x 20 x 10 In. on the 28th day after inoculation. at which time it was fluctuating and the ulcer had healed. On the 35th day after inoculation the swelling remained unaltered in size but now drained to the surface through a new sinus. By the thd 64 day the swelling had reduced to 1+0 1: 15 x 5 n. It continued to reduce thereafter. but the inoculation site was still detectable at necropsy. m gm. (56 days after inoculation) Holstein-Hereford steer. 9 months old. good condition. '000 lb. weight. The only lesion detected was at the site of inoculation. This consisted of a red nodule 5 ms. in diameter lying in the dermis and. adjacent to it. an abscess-like lesion 2 -. in diameter. These lq under the hairless area still visible but reduced to 5 mm. in diameter. mm M. Microscopic changes were limited to those seen at the skin injection site. The section through the abscess-like lesion showed a granuloma consisting of epithelioid and giant cells in which there was some central caseation and calcification. Acid-fast bacteria were rare. About the periphery of the granuloma some att-pt had been made at encapsulation. W W. Acid-fast bacteria were recovered from the left prescapular lymph node. the skin inoculation site. the pool of the anterior mediastinal and left and right bronchial lymph nodes. from the posterior mediastinal lymph node and from the pool of the liver and spleen . mum ma _C_a__lf g! - 1 mg. inoculu- intradermally. W W. The skin at the injection site never ulcerated. The swelling reached a maximum diameter of 30 u. on the 11th day after inoculation but regressed until on the 33rd day only a very slight swelling was detectable. 65 12.212222 {Ml-28b (71 days after inoculation) Holstein.heifer. 7 months old. good condition. 350 lb. weight. Ho gross changes were seen in the animal. The skin inoculation site was not detected. ‘fligtgpgthglggig’£in§ing_. Ho microscopic changes were found. flggtgziclcgicpgingiggg, Acid-fast bacteria were recovered from the left prescapular lymph node and the pool of the anterior and posterior mediastinal and left and right bronchial lymph nodes. Culture 19302-1 Q.l‘,3§_- 1 mg. inoculum intradermally. ‘glinisgl,2§gggygtigpg. 0n the 7th day after inoculation a circum- scribed firm swelling 25 mm. in diameter was seen at the site of inocue lation. A week later this had reached a.diemeter of 26 mm.. in which there was a central hairless zone 5 mm. in diameter. 0n the 21st day after inoculation the swelling had receded to 15 mm. in diameter. The central hairless area was still visible. but the lesion had ulcerated through this acne to the surface and was now covered by a scab. By the 28th day after inoculation. the lesion had.reoeded to a healing ulcer 3 mm. in diameter. Hy the 35th day after inoculation there was little swelling. but a central hairless area b me. in diameter was still clearly visible. 0n the “and day after inoculation the lesion was represented by only a hairless area 10 mm. in diameter. ‘flfigggglz,{1ngingg, (5“ days after inoculation). Holstein heifer. 8 months old. good condition. “00 lb. weight. lo significant gross lesions were seen. The skin inoculation site lesion had disappeared except for the area of hairlessness. and. 66 on section. no lesion was visible intradexmally. W m. a section of skin through a hairless area at what was presumed to be the inoculation site showed a diffuse granulomatous infla-atory response in the papillary layer and extending into the reticular layer. his consisted chiefly of macrophages and epithelioid cells which had made some attempt to form giant cells. 1 few heterophils and some lymphocytes were visible throughout the granu- lcma. Ho caseation or calcification was seen. Acid-fast bacteria were rare and confined to the epithelioid cells. W m. Acid-fast bacteria were recovered from the 1m. prescapular lymph node. x... a. skin inoculation site. from the pool of the anterior mediastinal and left and right bronchial lymph nodes and from the posterior mediastinal lymph node. 99; 31 - 1 mg. inoculum intradermally. M W. The inflammatory response at the skin inocu- lation site was similar to that seen with calf 32. ineculated with the“ eame organism. m the 7th day after inoculation. the swelling had reached a diameter of 25 mm. . was circumscribed but little elevated. A -weeklaterthe swellinghadreducedtolSmm. indiameter. againwas little elevated but had a central hairless red area 7 mm. in diameter, which had not ulcerated. m the 21st day after inoculation. the swelling had subsided but a central ulcer 5 -. in diameter was visible. This . was unchanged on the 28th day after inoculation. but on the 35th day only a hairless area 9 mm. in diameter remained. his was the only lesion seen also on the h2nd day. 6? W m. (51‘ days after inoculation), Holstein-Angus heifer. 9 months old. good condition. M0 lb. weight. Ho gross lesions were seen at necropsy. W W. No lesions were detected as the skin inoculation site could not be determined at necropsy and thus microscopic examination of the area was not feasible. W m. Acid-fast bacteria were recovered from the left prescapular lymph node. from the skin inoculation site. from the pool of the anterior mediastinal and left and right bronchial lymph nodes. from the posterior mediastinal lymph node and from the pool of the liver and spleen. mm now. an, 31 - 1 mg. inoculum intradermally. M obgengtiogs. Calf 31+ was killed in m on the 43rd day after inoculation because of what was later found to be bilateral pyelonephritis . he skin at the inoculation site never ulcerated. a diffuse intrsdermal swelling 10 n. in diameter developed at. the site of inoculation on the 7th dq after inoculation. his became mre circumscribedbythelhthdayandremainedasanodulelOm in diameter until the time of death. a: the lard day after inoculation the animal was noticed to be sick. It could not rise. was obviously . becoming comatose. and was killed in m. m M. (143 days after inoculation) Holstein steer. 9 months old. fair condition. 350 lb. weight. he gross lesions were confined to the kidneys. urinary bladder and the skin at the site of inoculation. The kidneys were 68 grossly swollen and. on the cut surface were mottled yellow and red with nuaerous streaks extending from the cortex to the medulla. The bladder was moderately distended with cloudy urine. The skin at the inoculation site showed a focus of yellow pus-like material 6 u. in diameter x 3 mm. thick. W W. it the skin inoculation site a granuloea- tous focus was present in the reticular layer of the skin. his consisted chiefly of epithelioid cells which showed some attempt to form giant cells.. Ho caseation nor calcification was seen. but an attempt had been made to encapsulate the lesion. Acid-fast bacteria were not seen. Both kidneys were involved with acute pyelonephritis. characterised by exten- sive cellular. and hyaline casts and an interstitial infiltration of leukocytes . W W. Acid-fast bacteria were reoowered from the left prescapular lymph node. from the skin inoculation site. from the pool of the anterior mediastinal and left and right bronchial lymph nodes and from the pool of the liver and spleen. fl Jé - 1 mg. inoculum intradermally. M W m the 7th day after inoculation a diffusely thickened intradermal swelling 10 n. in diameter was seen at the site of inoculation. A week later this was. still 10 I. in diameter but was more circumscribed. (h the 21st day after inoculation the swelling had reduced to a nodule 5 nm. in diameter. A soft intradermal nodule 8 mm. in diameter was present on the 30th day after inoculation. and a week later a hard nodule 10 mo. in diameter was there. which remained until necropsy on the 56th day. 69 .m m. (56 days after inoculation) Holstein steer. 9 months old. good condition. 600 lb. weight. The lesion at the skin inoculation site was the only one seen. This consisted of an abscess-like lesion 6mm. in diameter lying in the dermis md filled with yellow pus-like material. W; W. Microscopic examination of the skin inomlation site revealed an intradermal granuloma lying in the reticular lqer. This was circumscribed. with a central area of caseation and calcification which was surrounded by an epithelioid cell sons. The periphery of this sons was infiltrated by lymphocytes. Ho acid-fast bacteria were detected. W W. Acid-fast bacteria were recovered from the left prescapular. lymph node. from the skin inoculation site. from the pool of the anterior nediastinal and left and right bronchial lymph nodes. from the posterior mediastinal lymph node and from the pool of the liver and spleen. Culture 167C; -1 m; 3.1- 1 mg. inoculum intradermally. W W Seven days after inoculation theswelling at the inoculation site reached a diameter of 25 n. with a diffuse thickening of the skin. a week later the swelling was still 25 n. in diameterbutwasnchmm. inthickneas. anda5-.u1oerhadformed inthecenter. 0nthe21stdqtheswellinghadrecededtoljmn. in diameter and 5 -. in thickness: the ulcer was healing and had receded to h -. in diameter. m the 30th day after inoculation the skin inocu- lation site was indicated by a hairless area 7 mm. in diameter with 70 little swelling. but on the 37th day after inoculation the swelling recurred. reaching 35 u. in diameter and 5 n. in thickneswith a central hairless area 10 n. in diameter. By the h7th do after inocula- tion an 8 x 5 -. nodule had formed at the inoculation site. and lay under the hairless area of the skin. then 5 u. in diameter. . m m. (51’ due after inoculation) Holstein steer. 9 months old. fair condition. 650 lb. weight. The only lesion detected was that at the skin inoculation site. and this consisted of a somewhat diffuse 15 mm. intradermal thickening. W We... Himflmun ohms-s were «on only at th- skin inoculation site. his showed small foci of granulomatous inflamma- tory cells in the papillary and reticular layers of the skin. Macrophages with some lymphocytes were involved. but giant cells and acid-fast bacteria were not seen. There was no attempt at encapsulation. W m. Acid-fast bacteria were recovered fron the left prescapular lynph node. from the skin inoculation. site. from the pool of the anterior mediastinal and left and right bronchial lymph nodes. from the posterior mediastinal lymph node ad from the bone marrow from the left and right humeri. E5 27 - 1 mg. inoculum intradermally. M W. m the 7th day after inoculation the skin at the inoculation site showed a diffusely reddened and thickened area 15 u. in diameter with a central soft focus which had not ulcerated to the surface. Seven days later the swelling was 20 mm. in diameter with a central hairless area 5 ml. in diameter. By the let dq an 71 ulcer had developed which was 3 mm. in diameter and extruded a yellow caseous material. The swelling was l5’mm. in diameter and 5 mm. thick. (h the 30th day after inoculation there was no swelling. only a central hairless area 1! mi. in diameter. The lesion was unchanged on the 37th day after inoculation. but by the b7th dq the hairless area had been reduced to 7 mm. in diameter. 332122.91 my (56 am after inoculation) Holstein steer. 9 months old. good coalition. 550 lb. weight. Apart from the small hairless area at the site of injection. no gross lesions were detected. A cross section of the skin at the inoculation site failed to reveal an gross abnormality. W W. Microscopic examination of the skin at the site of inoculation showed several small microscopic foci of granu- lomatous inflammation lying in the reticular layer of the skin. These granulomatous foci consisted of macrophages and lymphocytes. Giant cells. caseation. calcification and acid-fast bacilli were not seen. W m. Acid-fast bacteria were recovered from the left prescapular lymph node. the pool of the anterior mediastinal and left and right bronchial lymph nodes. from the posterior mediastinal lymph node and from the pool of the liver and spleen. Group III lbcobacteria of Food and Soil Origin Five calves were inoculated with three cultures of Group III qcobacteria of feed or soil origin as follows: 72 mlture Number Calf Nulber feed origin Calves '46. #7 soil origin 131 Calf 48 X14]. Calves #9. 50 A 22322 at ma m c_al_£ 36; - 1 mg. inoculum intradermally. mm. m the 7thday after inoculation the swelling about the inoculation site had reached a diameter of 10 n. with an elevation of 5 -.. and in the middle of the elevation was a hairless area5u. indiameter. Onthelhthdqthe swellingwasalmostunchanged. being 10 mm.. in diameter and 1b n. in elevation. he cmitrel hairless areahadshrunkto 2-. indiameter. Bythe22nddqafterinoculation the injection site was represented by a nodule 5 n. in diameter. of which the central 3 me. was hairless. on on. 39th day after inoculation this nodule had receded to 2 n. in diameter. m m. (65 due after inoculation) Angus-Holstein crossbred bull. 11 months old. good condition. 500 lb. weight. Ho gross lesions were found. The exact skin inoculation site . could not be detected with certainty. W Ma- No 10810!!- were detected. W m Acid-fest bacteria were recovered from the left prescapular lymph node. the pool of. the anterior .diastinal and left and right bronchial lymph nodes and from the posterior mediastinal w ”d.e 73 ga_1_f EL - 10 mg. inoculum intradermally.. m W. (hi the 7th day after inoculation the swel- ling at the skin inoculation site had reached a diameter of 1“ -. with a 10 -. elevation. There was no ulceration and no hair. loss. By the lhth day the swelling was. 25 x 20 n. in area with an elevation of 3 n. and with a 2 x 7 mm. hairless area in the middle. light cum later tho swelling covered an area- of 35 x 25 n. with an elevation of 7 mm.. and in it lay two 5 mm.. indiameter ulcers. 0n the 28th day after inocula- tion the two ulcers. now 7 mm. in diameter. were still present. Ho - swelling ruained on the 39th day after inoculation. but two hairless areas 5 am. in diameter were visible. m m. (6“ days after inoculation) Angus-Holstein crossbred heifer. 9 months old. good condition. ’400 lb. weight. The only gross lesion seen was in the skin at the site of inoculation. Two areas. each approximately 10 n. in diameter. were slightly swollen. (hi cross section one of these was found to have a reddened ....5... indiameterandme. highinthereticular moi-or the skin. but no lesion was detected on cross section of the other. W W. he only microscopic change found was in the skin at the site of injection. In the reticular layer there was a patchy mononuclear cell infiltrate about the large blood vessels. Ho true granuloma was found. W W. . Acid-fast bacteria were recovered from the skin inoculation site. from the left prescapular lymph node. the pool of the anterior mediastinal and the left and right bronchial lymph nodes. 7b the posterior mediastinal node. the pool of the liver and spleen and from the bone marrow from each humerus. mfimm 131 m 58 - 1 mg. inoculum intradermally. M W. 0n the 7th dq after inoculation. a 5 m. nodule was found at the site of injection. Seven days later no lesion was detectable at the inoculation site. but on the 22nd day after inocu- lation there was a slight intradermal thickening but no hair loss. On the 28th and 39th due after inoculation no abnormality was found at the injection site. W m. (63 days after inoculation) Angus-Holstein crossbred bull. 10 months old. good condition. 600 lb. weight. Ho abnormality was detected. W W. No microscOpic changes were seen in any organs or lymph nodes. W m. Acid-fast bacteria were recovered from the left prescapular lymph node. and the pool of the anterior mediastinal and left and right bronchial lymph nodes. 116-1 9.9.1 32 - 10 mg. inoculum intradermally. W W. 01 the 7th dc after inoculation. the swel- ling at the site of the injection had reached a diameter of 35 mm. with a 5 mm. elevation. Ho hair loss had occurred. Seven days later the 75 swelling had receded to 10 mm. in diameter with an elevation of 2 mm.. but the central portion of the swelling had .a hairless area 3 mm. in diameter. By the 22nd day after inoculation the swelling had completely resolved emi the lesion consisted only of the area 5 n. in diameter which had lost hair. (hi the 39th day the lesion was similar to that seen on the 22nd day after inoculation. in that the lesion showed only a hairless area 5 mm. in diameter. m M. (73 days after inoculation) Holstein-Angus heifer. 10 months old. good condition. 600 lb. weight. Ho gross lesions were found. W W. Ho microscopic lesions were detected in am organs or lymph nodes. W W. Acid-fast bacteria were recovered from the left prescapular lymph nodes. from the pool of the anterior mediastinal . and left and right bronchial lymph nodes and from the posterior mediasti- nal lymph node. w 59 - 1 mg. inoculum intradermally. m W. 011 the 7th day after inoculation the swel. ling at the injection site reached a diameter of 10 n. It was hard but had little elevation. Seven days later the injection site was represented by an intradermal nodule 5 mm. in diameter with an elevation of 2 -. here was no hair loss. at the 22nd and 29th days after inoculation. the lesion was unchanged from that seen at lb days. 76 W m. (73 We after inoculation) Holstein bull. 11 months old. good condition. 550 lb. weight. Ho gross lesions were found. W gm. Ho microscopic changes were detected in my organs or lymph nodes. W W. Acid-fast bacteria were recovered from the lung and the posterior mediastinal lymph node. Pseudochromes Seven calves were inoculated with four cultures of pseudochrome mycobacteria as follows: Culture Number Calf Humber 528-1 Calves ll. 57. 58 613-0 all". no 55 1123-0 Calf 17 128F-0 Calf 56 Mm jail m u - 1 mg. inoculum intradermally. m W. The site of inoculation became swollen and reached a maximm diameter of 12 I. on the l#th dq after inoculation. It then subsided to be replaced by a hard nodule 5 u. in diameter.. No other clinical signs were noted. W W. (72 days after inoculation). Holstein steer. 9 months old. good condition. 350 lb. weight. mes lesions consisted of an intradermal nodule 10 x 5 mm. at the inoculation site. fibrous adhesions between the visceral and parietal pleurae over the diaphrapatic lobes of the lungs. and several encapsulated abscesses containing yellow-green pus in the substance of 77 the two diaphragmatic lobes. hose thoracic lesions were undoubtedly associated with calf pneumonia present in this group of experimental animals before they were infected with acid-fast bacteria. W m. Microscopic examination of the skin inoculation site showed a large focus of lymphocytes in the reticular lqer of the skin. but no granuloma was seen. The diaphragmatic lobes exhibited an acute bronchopnelusonia with pus cells lying in the alveoli and distention at the alveolar septa by inflammatory exudate. including serum. fibrin and some heterophils. Ho pathologic changes were detected elsewhere. Mg m. Acid-fast bacteria were recovered from only the skin inoculation site. an 52 - 10 mg. inoculum: 2 mg. each by the intradermal. subcutaneous. intraperitoneal. intramuscular and peroral routes. M W. The swelling at the site of the intredermal inoculation reached a maximum size of 10 a. diameter on the 15th day after inoculation. At the same time. a nodule 2 mm. in diameter developed at the intramuscular injection site and a diffuse swelling approximately 20 mm. in .diameter around the site of the subcutaneous injection. At necropsy all swellings had disappeared except for a very small nodule at the intradermal injection. site. None of these lesions ulcerated. Inter- mittent attacks of coughing were noted. presumably due to the inhalation of an aerosol of HT-383. which occurred on the day of inoculation. 78 W W. (58 days after inoculation) Holstein steer. 8 mnths old. good condition. 1550 lb. weight. Generalised pulmonary atelectasis was present in the right apical lobe and scattered linear areas of atelectasis were present in the right cardiac and intermediate lobes. he intradermal injection site was represented by a red area 1 am. in diameter in the dermis. W.Wo Skin at the inoculation site. A noncaseous granulue was found in the reticular layer of the skin. his consisted of epithelioid cells an! giant cells: acid-fast bacteria appeared to be absent. Although the granuloma was sharply delineated by the surrouMing fibrous tissue of the reticular lusr. there was no true attempt at encapsulation. Lung. An extensive chronic bronchiolitis was present throughout the section. Extensive fibrosis had taken place in the lamina propria of the bronchioles and bronchi and the adjacent alveoli had become atelectatic. W W Acid-fast organisms were isolated from posterior mediastinal nodes ooh. mfi-lOmg. inoculum: 2mg. eachbythe peroral. intradermal. subcutaneous. intramscular. and intraperitoneal routes. his organism was the roisolant recovered from the skin inoculation site of calf 11. W W. An intradermal nodule 10 mm. in diameter developed at the site of intradermal inoculation. his nodule never ulcerated. regressed slowly and had completely disappeared at the time of necropsy. Some slight thickening of the skin and subcutis was 79 noticed about the subcutaneous and intramuscular injection sites 21 days after inoculation. Intermittent attacks of coughing were present through- out the period of the experiment. induced. premably. by the inhalation of an IT-383 aerosol on the day of inoculation. mm (57 do: after inoculation). Holstein steer. 7 months old. good condition. 500 lb. weight. Ho abnormality was detected. he inoculation sites could not b. tome new new. a. 1mm vor- ...... W m. Acid-fast organisms were isolated on a single tube each from the posbrior mediastinal nodes and the pool of the left and right prescapular lymph nodes. m $3.29. “I 15 - 1 mg. inoculum intradermally. W W. he site of inoculation reached a maximum swelling of 25 u. in diameter 11 days after inoculation. then subsided and never ulcerated. No other clinical changes were seen. lam W (78 days after inoculation) Holstein heifer. 8 months old. good condition. M0 lb. weight. Ho gross lesions were seen. W W. Sections of the diaphragmatic lobes of the lung had a perihronchial follicular lymphoid hyperplasia and a purulent bronchcpneumcnia in some of the alveoli. 80 W W. Acid-fast. organisms were recovered from the left prescapular lymph node and the pool of the anterior and posterior mediastinal. and left and right bronchial lymph nodes. Sal: .51 " 1° ‘8: inoculum: 2 l8- Oach by the peroral. intradermal. sub- cutaneous . intramuscular and intraperitoneal routes. M We- The swelling at the intradermal inoculation site reached a maximum di-eter of 15 ms. 10 days after inoculation. It never ulcerated. and the swelling slowly subsided. Hodules 2-3 In. in diameter were formed at the sites of the intramuscular and subcutaneoous injection. being most prominent 16 days after inoculation. hese lesions completely resolved. at the day of inoculation the animal was eXposed to an aerosol of HT-383 and developed a moist cough which persisted throughout the experiment . . W W. (57 days after inoculation) Holstein-Angus crossbred steer. 8 months old. good condition. 900 lb. weight. Gross lesions were seen only at the site of the intradermal injection. This consisted of an intradermal red focus 1! mm. in diameter. W M. A tuberculous granuloma was present at the site of the intradermal inoculation. Giant cells. epithelioid cells. lymphocytes and numerous acid-fast organisms were present. W W. Acid-fast organisms were isolated from the left and right prescapular. posterior mediastinal and thoracic modes and liver-spleen pool. 81 Salim 1.22:2 Calif, 12 - 1 mg. inoculum intradermally. W W. The intradermal inoculation site reached a maximum swelling of 22 use. diameter on the 22nd day after inoculation. regressed and finally disappeared. limos: im- (80 do: after inoculation) Holstein heifer. 9 months old. good condition. 400 lb. weight. Ho gross lesions were seen. W W. No microscopic changes were detected in any organs or lymph nodes. W9, W. No acid-fast organisms were recovered. Salim £31.22. 93;; i6. - 10 mg. inoculum: 2 mg. each by the peroral. intradermal. sub- cutaneous. intramuscular and intraperitoneal routes. M W. A nodule 7 mm. in dimseter developed at the site of intradermal inoculation. here was no response to the subcutane- ous injection. but a nodule 2 am. in diameter developed at the intramuscu- lar injection site. None of these lesions ulcerated. and at necropsy no nodules remained. he steer had occasional attacks of coughing associ- . ated with the inhalation of an aerosol of HT-383 to which it was exposed on the day of inoculation. m gm. (56 days after inoculation) Holstein steer. 7 months old. good condition. 500 lb. weight. No gross lesions were found. he exact sites of injection were not ascertained. 82 W We Follicular hyperplane 01’ th- perihron- chial lymph nodes was present. here was a subacute tracheitis with heterophils in the epithelium. epithelial hyperplasia and subepithelial mononuclear infiltration. W W. Acid-fast organisms were isolated on one tube each from posterior mediastinal nodes and lung. and two tubes from the liver-spleen pool. Group IV wcobacteria Twelve calves were inoculated with six cultures of Group IV syco- bacteria as follows: Culture Humber Calf lumber lZlHP-O Calf 5 “17-1 Calf 9 375-0 Calves 12. 22 1173-0 Calves 15. 28. 52 71-1 - Calves 18. 51 ZSW-l “17.3 259 530 5’" Shim 222:9. 9.9.]; 5 - 2.2 mg. inoculum intradermally. W 2mm- None tumble- m We (37 days after inoculation) Holstein. heifer. 8 months old. Ho gross lesions were detected. W m. A few scattered lung lobules show“ atelectasis. W m. Acid-fast bacteria were recovered from the pool of the anterior and posterior mediastinal and left and right bronchial lymph nodes . 83 2:23am 592:1. 95; 2 - 1 mg. inoculum intradermally. W W. he intradermal inoculation site showed a maximum swelling of 18 m. diameter 1% days after inoculation. It did not ulcerate: rather. it subsided and at necropsy could not be detected. W W. (69 days after inoculation) Holstein steer. 9 months old. good condition. 500 lb. weight. Ho gross lesions were detected. W m. Ho abnormality was detected. W m. Acid-fast bacteria were recovered from the left prescapular lymph node. mm 518:2 m: 13. - 1 mg. inoculum intradermally. M W. he week after inoculation the intradermal inoculation site showed a swelling 5 ms. in diameter. The animal was recumbent and unable to rise. and it died the next dq. W m. (8 days after inoculation) Breed and sex not recorded. 7 months old. fair condition. weight unknoim. . he cause of death was not determined. W W. ally the skin inoculation site and the left prescapular lymph node were examined. Ho significant change was seen in the lymph node. but a small granuloma was. found in the reticular layer of the skin. his consisted of macrophages and lymphocytes: giant cells were not seen. Acid-fast bacteria were very numerous and especially so at the periphery of the granuloma. 8} W 2%. Due to the post-mortem decomposition no bacteriological examination was attempted. £51; _2_2 - 1 mg. inoculum intradermally. M W. No inflammation was seen at the site of the intradermal injection. Twenty-eight days after inoculation the animal was found dead in its stall. W W (28 days after inoculation) Holstein steer. fair condition. Early pneumonic changes were present throughout the lungs. he skin inoculation site could not be detected. W my» 0&1: the loft pro-«paler lynph node. liver. intestine. kidneys and lungs were examined. he liver showed a generalised centrolobular degeneration with hemorrhage into these areas. Iarly purulent bronchcpneumonic changes were seen in the lungs. vis.: hyperemia. pus cells and debris in the bronchioles and alveoli and infla-atory edema and heterophils in the interlobular septa. W m... Ho acid-fast bacteria were isolated. mm 222:9 Elli _l§_ - 1 mg. inoculum intradermally. w W. A swelling 20 a. in diameter was present at the site of inoculation 7 days after inoculation. his subsided over the next fortnight and them completely disappeared so that the inoculation site was not detectable at necropsy. 85 W m. (78 days after inoculation) Holstein heifer. 8 months old. good condition. No lb. weight. Ho gross lesions were detected. W m. Ho lesions were seen in any organ or lymph node. W3 gimme. Ho acid-fast bacteria were recovered. 2a}; 28 - 1 mg. inoculum (killed) intradermally. W W Fourteen days after inoculation an intra- dermal nodule 2 mm. in diameter developed at the site of inoculation. his disappeared in the following week. Ban-am Simian. (70 day- after inoculation) Holstein-Angus crossbred heifer. 7 months old. good condition. #50 lb. weight. Io gross lesions were detected. We union. no i..i... were detected- W M. No acid-fast bacteria were recovered. M 53 - 10 mg. inoculum: 2 mg. each by the peroral. intradermal. sub- cutaneous. intramuscular and intraperitoneal routes. W W. he swelling about the intradermal inocula- tion site reached a maximum diameter of 10 -. nine days after inoculation. his slowly subsided and at necropsy the injection site could not be found. He infla-atory response was seen at am peried about the sites of intramuscular. intraperitoneal and subcutaneous inoculation. his Figure 17. Left prescapular lymph node from calf 7. inocu- lated with 710-0. a Group III mycobacterium. Note the necrotic cell debris (l) and the epithelioid cells (2) among lymphocytes lining the internal surface of the cap- 81110. N." ugh.“ ' H & to x187e Figure 18. Skin inoculation site from calf 52. inoculated with 1173-0. a Group IV mycobacterium. Note the diffuse mononuclear clear cell infiltrate in both the reticular and papillary layers of the skin. New Fuchsia - H a 8. x75. 87 animal was also exposed to an aerosol of HT-383 on the day of inoculation and subsequently showed intermittent attacks of coughing. 1112:2211 m. (52 days after inoculation) Holstein heifer. 7 months old. fair condition. 550 lb. weight. Ho gross lesions were detected. fligtgpgthglggig,£ig§iggg. .L section through what was thought to be the inoculation site showed mononuclear cells infiltrating diffusely through the fibrous bundles of the reticular leyer of the skin (Figures 18 and 19). In the lung the bronchioles were affected with a subacute inflammation with excess goblet cells. infiltration of mononucleers into the lamina propria. epithelial hyperplasia and heterophil infiltration into the epithelium. Similar inflammatory changes were present in the trachea. [figgtgziglggig_1indipgg. Acid-fast bacteria were recovered only from the posterior mediastinal lymph nodes. comm-l 92%;.lé - 1 mg. inconlmm.intradermally. W W. No inflammatory response was seen at the inoculation site. Eggzgpgz,11nflipgg. (86 days after inoculation) Holstein steer. 10 months. good condition. #50 lb. weight. lo gross lesions were detected. Biltgngthglggighfiinflingg. Ho abnormality was detected in any organs or lymph nodes. ' A higher magnifi- cation to show the mononuclear cells in the reticular layer of the skin and the absence of giant cells and caseation. Skin inoculation site from calf 52. inoculated New Fuchsia - H d I. x187. with 1173-0. a Group IV woobacterium. Figure 19. his contains How Fuchsia - H a 8. Right axillary lymph node from calf 51. inocu- lated with 7F-l. a Group IV wcobacterium. a single noncaseous granuloma (arrow). x75. Figure 20. 89 W M. Acid-fast bacteria were recovered from the left prescapular lymph node. 3;; §_ - 10 mg. inoculum: 2 mg. each by the peroral. intradermal. subcu- taneous. intramuscular and intraperitoneal routes. W W. he swelling about the intradermal inocula- tion site reached a diameter of 20 .. nine days after inoculation. Six days later it was unchanged. but the subcutaneous injection site contained a nodule 30 mm. in diameter and the intramuscular site a swelling 20 ms. in diameter. These slowly regressed and at necropsy only the intradermal site could be detected. 0n the day of inoculation the animal was exposed to an aerosol of HT-383. and five days later it was coughing continuously with the expulsion of mucus. his clinical sign continued for approxi- mately three weeks. W m. (52 days after inoculation) Holstein steer. 8 months old. poor condition. l+50 lb. weight. The right apical and cardiac lobes were red and firm due to atelectasis. and their bronchioles had thickened walls. Skin inoculation site. An encapsulated "abscess." 10 mm. in diameter which contained cream yellow pus was present in the dermis. left prescapular lymph node. Several caseous food 1-2 a. in diameter were visible. Internal iliac lymph nodes. Humorous yellow foci varying in diameter from 0.5 to is m. were seen. fligtgpgthologig m. Skin inoculation site. Several granulomas (perhaps part of one granuloma) had central cessation in which were accumulations of heterophils. 90 These were surrounded by epithelioid cells in which were numerous giant cells. No acid-fast bacteria were found. There was no attempt at capsule formation. Left prescapular lymph node. A.noncaseous granuloma and another encapsulated granuloma.with central caseation and calcification were seen. Epithelioid cells and giant cells were numerous. Ho acid-fast bacteria were detected. Right axillary lymph node. Two noncaseous granulomas ley subcapsu- larly and consisted of epithelioid cells and few giant cells (Figures 20 and 21). Ho acid-fast bacteria were seen. Internal iliac lymph node. Multiple nonprogressive encapsulated granulomas with central caseation and calcification were seen (Figures 22 and 23). No acid-fast bacteria were detected. Right ischiatic lymph node. One noncaseous granuloaa of microscopic else was present and consisted of epithelioid cells and a few giant cells (Figure 2“). A giant cell contained a few acid-fast bacteria. hung. right apical lobe. Chronic bronchiolitis was present. with the epithelium.showing hyperplasia and infiltration with heterophils and the lamina propria heavily fibrosed. thy of the alveoli were atelectatic. Trachea. A subacute tracheitis was present. with epithelial hyper- plasia. excess goblet cells and infiltrating heterophils in the epithelium and lamina propria. W W. Acid-fast bacteria were recovered from the skin inoculation site. the left prescapular lymph.node. the pool of the anterior mediastinal and left and right bronchial lymph nodes. from the posterior mediastinal lymph nodes. the pool of the liver and spleen and from the internal iliac lymph nodes. Figure 21. Right axillary lymph node from calf 51. inocu- lated with 7F-l. a Group IV Ivcobacterium. This higher magnification of a portion of Figure 20 shows the epitheli- oid cells in the center of the noncaseous granuloma. New meh'in - H & Be 8187e Lh Figure 22. Internal iliac lymph node from calf 51. inocu- lated with 7F-l. a Group IV mycobacterium. An encapsulated caseo-calcareous granuloma lies under the capsule. New Fuchsin - H a 3. x50. . Figure 23. Internal iliac lymph node from calf 51. inocu- lated with 7F-1. a Group IV myccbacterium. Granuloma is seen with central calcification (l). a layer of caseation (2). a layer of epithelioid cells and giant cells (3). all enclosed within a fibrous capsule (1.), New Fuchsia - H a I. x187. Figure 215. Right ischiatic lymph node from calf 51. inocu- lated with 7F-l. a Group IV mycobacterium. A solitary noncaseous granuloma with two giant cells lies in the field. New Fuchsin - a a a. x75. ' 93 mm 2222.1. §_a_lf_2§_- 1 mg. inoculum intradermally. M W. Sixteen days after inoculation the injection site opened and discharged a yellow pus-like material and then heeled over completely in 10 days. 19212221 m. (70 days after inoculation) Holstein bull. 6 months old. good condition. 1:00 lb. weight. The only gross lesions detected were a hard red module 1 u. in diameter in the dermis at the site of inoculation and scattered lobules of red consolidation on the ventral border of the right intermediate lobe of the lung. W m. Because of the small sise of the skin lesion this material was used solely for bacteriological examination. W W. Acid-fast bacteria were recovered from the left prescapular and the pool of the anterior and posterior mediastinal and left and right bronchial lymph nodes. Calf 52 - 10 mg. inoculum: 2 mg. each by the peroral. intradermal. subcu- taneous . intramusmlar and intraperitoneal routes. W W. he swelling about the intradermal inocula- tion site reached a maximum diameter .of 10 mm. at nine days after inocu- lation. raained at this size for 20 days and then slowly subsided so that the inoculation site was not detected at necropsy. No infla-atory response to the intraperitoneal and intramuscular injections was seen. At the subcutaneous inoculation site a closed-in nodule 20 mm. in diameter had developed by the 15th day after inoculation and then subsided. No 91b ulceration occurred at any of the inoculation sites. This animal was also exposed to an aerosol of ET-383 on the day of inoculation but was never seen to cough. m M. (57 days after inoculation) Hereford-Holstein crossbred steer. 8 months old. good condition. 800 lb. weight. he only gross lesions seen were extensive atelectasis of both the apical and cardiac lobes of the right lung. W m. he lesions seen were confined to the respiratory system and consisted of a subacute tracheitis. bronchitis and bronchiolitis. and patchy atelectasis. The infla-atory changes in the respiratory passages were characterised by epithelial hyperplasia. excess goblet cells. mononuclear infiltration of the lamina propria and the migration of heterophils through the epithelium. W m. Acid-fast organisms were isolated from the liver-spleen pool. lung. and from the pool of the anterior mediastinal and left and right bronchial lymph nodes. the posterior mediastinal nodes and the pool of the left and right prescapular lymph nodes. galf fl - 10 mg. inoculum: 2 mg. each by the peroral. intradermal. subcu- taneous. intramuscular and intraperitoneal routes. his organism was roisolated from calf 25. m W. Nine days after inoculation the intradermal inoculation site presented a closed module 10 mm. in diameter. his reached a maximum diameter of 20 I. 15 days after inoculation. remained unchanged for a further week and thereafter slowly receded to 15 -. in 95 diameter at time of necropsy. he submitaneous and intramuscular inocula- tions cansed diffuse acute inflammatory responses 30 n. in diameter 15 due after inoculation. A week later these had receded to 20 n. in diameter: 28 days after inoculation the subcutaneous inoculation site had only a scar and the intramuscular site had a slightly elevated swelling 10 n. in diameter. No response to the intraperitoneal injec- tion was seen. No ulceration occurred at am site of inoculation. his animal was exposed to an aerosol of ET-383 on the day of inoculation and subsequently showed only an occasional attack of coughing. although a rapid respiration rate was seen three to four weeks after inoculation. W W. (53 days after inoculation) Hereford-Holstein heifer. 7 months old. good condition. 800 lb. ".mte The skin at the site of intradermal inoculation contained an encapsulated abscess 10 mm. in diameter. in which was a yellowish granular "pm". No other lesion was seen. W m. he skin contained one discrete granuloma about which some attempt at encapsulation had been made. Acid-fast bacteria were readily visible in a giant cell. Kidney. Several microscopic foci of interstitial nephritis con- sisting of periglomerular fibrosis and interstitial infiltrations of mononuclear cells were seen. Liver. Activation of the Iupffer cells had occurred. as judged by their prominence due to swelling. Several scattered microscopic foci of mononuclear cells 147 in many of the lobules. )bnonuclears and hetero- phils were present around the components of may of the portal triads. 96 , bachea. bronchi. lungs. Subacute infla-atory changes of the air passages and some atelectasis were seen. the lesions being similar to those in calf 51. W W. Acid-fast bacteria were recovered from the skin inoculation site. the left prescapular lymph node. the pool of the anterior mediastinal and left and right bronchial lymph nodes. the posterior mediastinal. the lung and the pool of the liver and spleen. 21- 23.1.2: One calf was injected with live h- m and another with killed a. m. 1 mg. organisms being injected intradermally into the skin of the lateral surface of the left foreleg just above the carpus. La1_f_ _2_l - 1 mg. inoculum (n. m) intradermally. m W. Seven days after inoculation a swelling 30 mm. in diameter had developed at the site of injection. his had receded to 25 mm. on the 14th day after inoculation and remained unchanged on the 23rd day. (hi the 31st day the swelling was 33 mm. in diameter: six days later it reached the diameter of 1&5 ma. . was soft and fluctua- ting and had an elevation of 10 mm. By the 52nd day after inoculation the swelling had returned to the diameter of 33 mm. and remained this else as fluctuating swelling until necropsy. them We. 18“ days after inoculation) Holstein heifer. 9 months old. good condition. 450 lb. weight. he only gross lesions found were in the skin and the subcutis adjacent to the site of inoculation. 0n cross section of the skin a #0 x 30 x 20 mm. abscess. filled with a cream pus. was found lying in 97 , the dermis. In the subcutis under this lesion was another abscess which measured 35 x 25 x 15 mm. and was also enclosed by fibrous capsule. gistopathologic finding . The only lesions detected were those in the skin and the subcutis at the site of inoculation. These consisted of. a central caseous mass surrounded by a layer of epithelioid cells. Lympho- cytes were scattered between the macrophages and there were occasional foci of heterophils also. Acid-fast bacteria were readily visible within the epithelioid cells. Early calcification had taken place in the caseous mass. Lymphocytes were most concentrated at the periphery of the granu- loma directly under the fibrous capsule which completely enclosed the lesion. Esoteliologig finding . Acid-fast organisms were reisolated from the pool of the anterior and posterior mediastinal and the left and right bronchial lymph nodes and from the left prescapular lymph node. £95123] - 1 mg. inoculum (killed 2;. 9.11%) intradermally. Clinical Obsegzgtion . The only response to the intradermal inocu- lation was the development of a firm nodule. On the 14th day after inoculation a circumscribed intradermal nodule 10 mm. in diameter had formed. Seven days later this had reached a diameter of 15 mm.. but by the 28th day it had regressed to’a diameter of 8 mm. A diffuse intra- dermal thickening 5 nm. in diameter was the only evidence at the injection site on the 35th dry after inoculation. and this persisted until necropsy on the 56th day. figs;gggy;§ingiggg. (56 days after inoculation) Hereford-Holstein heifer, 8 months old. good condition. #00 lb. weight. 98 Apart from a diffuse thickening of the skin at the site of the intradermal injection. no gross lesions were detected. W m. The only microscopic changes seen were in the skin at the site of the inoculation. Microscopic noncaseous grann- lomas were found lying in the papillary layer of the skin. mess consisted of macrophages with some fibroblasts. eosinophils and lymphocytes. There had been no attempt at encapsulation and no acid-fast bacteria were seen. W W. No acid-fast bacteria were recovered on bacteriological examination. s. m M calves. 6 and 20. were inoculated with live n. m. lumber 6 received 2.2 mg. intradermally. and number 20 received 1 mg. intra- dermally. Calf 26 was inoculated with 1 mg. of killed 3. m. Calves 6 and 20 developed acute tuberculosis and were killed when they were in m. Neither was tuberculin-tested. it necropsy both animals had widespread lesions of tuberculosis involving most of the lymph nodes and parenchymatous organs in the body. These lesions were highly pro- gressive and showed marked central caseation with some slight degree of calcification and with no effective development of peripheral capsules. g_a_l_f é - 2.2 mg. inoculum (1301-0. 5. M) intradermally. M obsmatigg. The animal was killed in m; on the 37th day after inoculation. W W. (37 dqs after inoculation) Holstein heifer. 6 months old. poor condition. Gross lesions were found in the following lymph nodes: 99 mesenteric. internal iliac. right deep inguinal. right prescapular. left prescapular. right popliteal. anterior and posterior mediastinal and left and right bronchial. Hiliary lesions were visible throughout the lungs. There were four sites of skin inoculation - two on the left foreleg and two on the right hindleg. 1 mg. and 0.1 mg. being injected into separate sites on both these legs. Upper skin inoculation site. left foreleg. A swelling 1&0 mm. in diameter with a 15 an. ulcer was visible. The ulcer had eroded through the epidermis and well into the dermis and was covered by a greenish necrotic exudate. There was a moderately developed fibrous capsule around the lesion. lower skin inoculation site. left foreleg. The swelling was 10 n. in diameter. and a 5 n. ulcer lay at its center. this was poorly encapsu- lated and contained a greenish-yellow. thick. necrotic debris. Lower skin inoculation site. right hind leg. in ulcer 20 u. in diameter. which was fairly well encapsulated and covered by a reddish- yellow emdate lay in the epidermis and dermis. Upper skin inoculation site. right hind leg. in ulcer 30 u. in diameter and covered with a scab was enclosed by a firm fibrous capsule and had a caseous necrotic center. W W. a: microscopic examination. all lymph . nodes with gross lesions were found to have extensive areas of caseation necrosis. which were commonly confluent. here was some degree of cal- cification. Peripheral to the caseo-calcareous lesions was the granu- lomatous infla-atory reaction. his bad mmerous epithelioid and Langhans' giant cells and acid-fast bacteria. were common. here was no effective encapsulation by fibrous tissue. 100 lung. The section examined showed generalised consolidation with very few alveoli containing air. Macrophages. heterophils and acid-fast bacteria were visible in the bronchioles. whose walls showed epithelial lurperplasia an! infiltration of the lamina propria with numerous mono- nuclears. The alveolar spaces were obliterated by edema fluid. congested alveolar walls. macrophages and alveolar septal cells. Extensive coagu- lation necrosis of some areas had taken place and acid-fast bacteria were evident at the periphery of these lesions. Kidney. Bo abnormality was detected. Spleen. Q) examination under the lower power objective no abnor- mality was seen. However. closer emination showed that in a few of the germinal centers there were focal collections of macrophages and adjacent to these were a few acid-fast bacteria. Another one of these noncaseous granulomas had a Lsnghans ' giant cell which contained acid-fast bacteria. W m. Acid-fast organisms were isolated from all tissues. g 32 - 1 mg. inoculum (81-0 swine origin 5. 33911;) intradermally. MW Seven days after inoculation a swelling l50mm. in diameter had developed at the site. Seven days later this had reached #5 mm. in diameter and was commencing to ulcerate. The swelling had readied a diameter of 55 n. on the 23rd daysfter inoculation and bythe 31stdqwas 50-. indiameterwithanopenulcer23mm. in diameter. By the 37th day after inoculation the swelling had further increased to a diameter of 60 an. and had an ulcer 25 I. in diameter and was healing and granulating. The left prescapular lymph node was still grossly enlarged. The swelling had increased to 70 mm. in diameter on the 53rd day after inoculation and the ulcer was then 30 mm. in lOl diameter. The left prescapular lymph node had further increased in size and now measured approximately 130 x 250 mm. Six days later the ulcer had granulated so that it was only 7 u. in diameter. The left prescapu- lar and the posterior cervical lymph nodes were grossly enlarged. The animal was rapidly losing condition. was coughing frequently and had rapid abdominal breathing. W W (65 days after inoculation) Holstein steer. 9 months old. poor condition. #00 lb. weight. Generalised lesions of tuberculosis were evident. the following lymph nodes showing gross lesions: anterior and posterior mediastinal. left and right bronchials. medial retropharyngeal. posterior cervical. left prescapular. left axillary. hepatic. and the mesenterics. in ulcer 15 mm. in diameter was evident at the skin inoculation site. Subcutaneous tissue underlying it was thickened up to 20 m. in thickness and contained numerous foci of caseous yellowish material 2-3 mm. in diameter. Liver. Numerous white foci measuring 3-4 mm. in diameter were scattered subcapsularly and throughout the parenchyma. Lung. Numerous miliary tubercles were scattered throughout the whole of the lungs and were most evident towmd the dorsal borders of the diaphragmatic surfaces of both the diaphragmatic lobes and in the left apical lobe. The typical focus. when cut in cross section. showed a central white area surrounded by a red periphery. niltopgthologig m Extensive lesions of progressive tuber- culcsis were found in all the lymph nodes listed above an! were essentially the same in all lymph nodes. The inflammatory change was characterised 102 by multiple foci of caseation (Figure 25) which had often become confluent. In many lymph nodes the normal architecture had been obliterated by caseous necrosis (Figure 26). and in the necrotic areas only the blood vessels and a few lymphoid cells immediately adjacent to them remained visible (Figure 27). Skin. The surface of the lesion was covered by necrotic debris (Figure 28). but multiple granulomas with central caseation and early calcification were present in the reticular layer. The caseous areas were surrounded by granulomatous tissue in which there were numerous lenghans' giant cells (Figure 29). No acid-fast bacteria were detected. Liver. While no macroscOpically visible granulomas were present. there were numerous microscopic ones scattered throughout the section. These varied from foci of several macrophages to larger granulomas con- taining giant cells and with early caseation. No acid-fast bacteria “1‘. 8.011e ‘flggfigziglggig,£1g§ingg. Acid-fast organisms were isolated from all tissues. Q‘;;.g§ - 1 mg. up hayig,(killed) intradermally. ‘Qliniggl,gbgg:1|§igng, By 1“ dmys after inoculation a firm intra- dermal nodule 3 mm. in diameter had developed at the site of inoculation. Seven days later this had increased to a diameter of 10 mm.. wes non- fluctuant and closed. By the 28th day it had reduced to 8 n. in diameter and remained this size until necropsy. Figure 25. Anterior mediastinal lymph node from calf 20. inoculated with n. bovig. Note the areas of caseation (l. 2) and the granulomatcus inflammatory tissue in which there are numerous giant cellsC».New Fuchsin - H i E. 875. Figure 26. left prescapular lymph node from calf 20. inocu- lated with g. bOVis. Under the lymph node capsule (1). there remains a thin rim of lymphoid tissue (2). heavily infiltrated by granulomatous tissue (3) which surrounds the massive area of caseous necrosis (b). New Fuchsin - H i B. 150 e 101i Figure 27. loft prescapular lymph node from calf' 20. inocu- lated with 5. Egg. Extensive areas of caseation (l). in which there are scattered islands (2. 3) of lymphoid tissue around blood vessels. New Nchsin - H a 8. 175. ~~_ _ A in” 7 Figure 28. Skin inoculation site from calf 20. inoculated with H- he s. The surface of the ulcer is covered'by necro- tic debris arrow). and under this is granulomatous tissue which extends through the skin into the subcutis. New Fuchsin - H a I. x50. Figure 29. Skin inoculation site from calf 20. inoculated with g. bovis. Note the caseous area (1) surrounded by granulomatous tissue (2) in which there are Langhans' giant celés (3) and scattered lymphocytes. New‘Fuchsin - H 8: 3. x1 7. ,7 M—rivv-v—u Figure 30. Skin imculaticn site from calf 26. inoculated with killed L1. bovis. Note the extensive infiltration of mononuclear cells in the reticular layer. New Fuchsin - N a 3. x187. . 106 m M. (54 days after inoculation) Holstein steer. 6 months old. good condition. 1‘50 lb. weight. The only lesion detected was a pale yellow focus 8 u. in diameter lying in the dermis. seen on cross section of the skin inocula- tion site. W m. Iticrosccpic changes were confined to the skin inoculation site. A focal granuloma consisting of macrophages and some lymphoid cells lay in the reticular layer (Figure 30). Acid-fast bacteria. giant cells. caseation and calcification were absent. 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Results of hematologic examination 5 of blood samples taken immediately prior to necropsy. go gmmfigbmni ) “was” - .EEESEQSIEt ..P°r.§£_._. 26 16.“ #6 6,500 27 12.2 not done not done 28 11.? not done not done 29 20.2 59 8,000 30 17.3 52 ' 11.500 31 16.7 57 6.150 32 19.6 50 9.200 . 33 19. 3 62 5 .100 34 16.7 49 5.750 35 17.3 50 8.500 36 17.2 55 6.900 37 15.3 49 8.“50 38 15.3 49 10.600 39 16.3 48 8.500 40 17.6 52 8.350 #1 9.7 30 10.200 #2 15.3 46 10.500 an 17.9 52 12,650 “5 17.6 ‘ 55 11.750 #6 16.3 52 11.100 47 19.6 56 5.550 GEE? Hemogfobin wt 0 G 1.3 “9 50 51 52 sh ‘ 100 .1.) 17.9 17.0 18.3 1h.3 12.? 1b.} 123 TABLE IV--C0nt1nued 53 #8 50 1+5 38 #1 leukocytes 7.400 8,000 11,200 16.000 7.800 8,300 TABLE V. 12h Summary of the development of skin lesions at the inoculation site in calves inoculated ‘lith Group III qycobacteria of bovine origin. Days after Granuloma Culture 031! Skin inoc. ulcer Max. at inoc. No. No. Ulcer appeared Dimensions site Lesions 510-0 2 N/A‘ ... ... yes progressive gen- eralised (killed)29 no ... ... yes 1/d only“l #2 yes 33 7 In. yes primary complex #3 yes Zh 15 mm. yes generalised 680-0 1+ N/A 0 e e e e e yes 80n0r1112d #5 yes 32 10 um. yes generalized 503-0 1 N/A ... ... N/A generalised 620-0 3 N/A ... ... yes primary complex M yes 23 10 mm. yes generalised 1073.0 13 no ... ... yes i/d only 40 no ... ... no NHL"* 710-0 7 yes 10 25 um. yes primary complex 38 yes 15 10 an. no NHL 39 no ... ... yes i/d only 788-0 10 no 0 e e e e e yO‘ i/d only 'N/A - data not available uNHL - no microscopic lesions *'*i/d - intradermal lesion 125 TABLE VI. Summary of the development of skin lesions at the inoculation site in calves inoculated with Group III mycobacteria of swine origin. Days after Grsniioma Culture Calf Skin inoc. ulcer Max. at inoc., c No es eared Dimensions site Lesions 93C-0 16 no ... ... yes primary complex 176Cl-1 23 no e e e e e e no NML‘ 30 yes 21 5 mm. yes i/d only“ 31 yes 21 5 mm. yes i/d only 1730-1 2“ no ... ... no NHL 19302-1 32 yes 21 5 mm. yes i/d 33 yes 21 5 mm. no NML 186C-1 3“ no e e e e e e yes i/d 36 no ... ... yes i/d 16701-1 35 yes 14 5 mm. yes i/d 37 yes 21 3 mm. yes i/d *NML - no microscopic lesions "i/d - intradermal lesion 126 TABLE VII. Summary of the development of skin lesions at the inoculation site in calves inoculated with Group III mycobacteria of feed and soil origin. Days efter GranEIoma Culture Calf Skin inoc. ulcer Mex. st inoc. No Ulcer a ed Dimensions site Lesions Feed origin 46 no ... ... no NML‘ #7 yes 22 5 mm. no i/du Soil origin “‘8 no e e e e e e no NHL “’9 no e e e e e e no NHL 50 no e e e e e e no NHL ‘NML - no microscopic lesions *‘i/d - intradermal lesion TABLE VIII. Summary of the development of skin lesions at the inoculation site in calves inoculeted with peeudochromesz' qus after Granulona Culture Celf Skin inoc. ulcer st. st inoc. 0 ed ensions s t Lesions 528.1 11 no e e e e e e no NHL“ 57 no ... ... yes NHL 58 no e e e e e e no N 618.0 1“ 110 e e e e e e no NHL 55 no ... ... yes i/d** 1128.0 17 no e e e e e e no NML Rat-0 56 no e e e e e e no NHL *NHL -.no microscopic lesions *‘i/d - intradermal lesion 127 TABLE IX. Summary of the development of skin lesions at the inoculation site in calves inoculated with Group IV'mycobacteria. , ' h— Days after 2" Granuloma Culture Calf Skin inoc. ulcer Mix. at inoc. o c e Dime sions sit Lesion; lzuF-o 5 N/M no m" 2"1 9 no eee eee no NHL 878.0 12 no eee eee yes 1/d.“ 22 no eee eee no NHL 1178.0 15 no eee eee no NHL 28 no eee eee no NHL 52 no ... ... no NHL 7Fbl 18 no ... ... no NHL 51 no ... ... yes primary complex 25fir-l 25 yes 16 3 mm. yes i/d 53 no eee eee no m 5h no ... ... yes 1/d I"BI/A - data not available "NML - no microscopic lesions "*i/d - intradermal lesion 128 TABLE I. Summary of the development of skin lesions at the inoculation site in calves inoculated mu. m man- an» Days after Culture Calf Skin inoc. ulcer Max. at inoc. Em Hg , Ulcer appeared Dimensions site 11. b_gg_i_._s_ 6 yes N/A‘ ‘ 20 mm. yes 5 m 20 yes 115 30 mm. Y" (killed)26 no .. . ... yes LL 9“ 21 no . . . . . . yes 2? no . . . . . . yes Granaoma gsiong generalised generalized 1/dee i/d i/d I'N/A - data not available "i/d - intradermal lesion .coaveonsa :«Hsoaensp scape no ova. one we peg» can» aeueeam .33 m vesea as as: gowns.so«aoen:« :«Hseneos» ceaaesmea no code env as eeenxodna max» :4 eeeoaonHe 3 1 1m a m r S 3 .38 an m H u o n o m a o N m o o o o o o o o n m sehauaeom name: we we encomeom .11 + - + ' + - + I + ”COanM «13 a s 33 no cosh >H among .esoauoa an“: uuaseeu veep cannoneosu Heofisneo e>aueasdeoe mo soaosaeuaoo .HHN mamas on wmw m m 0H 0H, madcap ma 3 c w o o o H H m 0 one: ea N H o o n o m o o a .3: sum m H o H o o o m o o 3 .35 e \H mason mm as omnoauom - ..u - + ' + - + ' + .ugdu. 30H «duo. um o..HO 00 10.. 0 . Oh . O >4 .3..u.s.. .. ., 5..5. no mesh pm cacao HHH cacao .33...” .3: «.38... £3 finesse» 38 188 no 8338.30 .Hx Sm: TIBLE XIII. 130 Sunny of rosults of tuberculin tests on oslvos injocted with killed woohsotoris Cmdh' Fold rut. Increase in am. Mon. Response on Cor-- 9.11“ N . M st 48 hm; neg; Inst 26 H0 m 2 Msmdian and avian oquol st 1&8 hours 27 H- m 5 Johnin at 1+8 hours 28 1173-0 1.5 Johnin at 21+ hours Group IV 29 510-0 Group III 0 Johnin at. 1&8 hours ...- V. DISCUSSION Evaluation of Pathogenicity of Atypical Wcobacteria The data on animals inoculated with Group III qcobacteria are pre- sented in TABLE II. m In W 2: mm: m . Analysis of the results shows that Group III qcobacteria of bovine origin produced the whole range of lesions froa snall. barely visible granule-stun foci at the skin inoculation site to generalized progressive lesions. Seven cultures. or their reisolants. were inoculated into lb. calves - Nos. 1. 2. 3. l}. 7. 10, 13, 29. 38, 39, 40, and 1+2 through 50. Six calves (Nos. 1, 2. l}. 1&3. M, #5) had generalized disease. three (Nos. 3. 7. 1+2) had lesions in the lymph nodes draining the inoculation sites, three had lesions at the inoculation site and two had no gross or nicroscOpic lesions. Calf 29 was injected with a killed culture. Even though generalized disease was produced in 6 calves. the lesions were not as progressive as those seen in calves 6 and 20. inoculated with 5. 12211.!- Only three of the six (Nos. 2. 3, and 155) were classed as progressive: Nos. 43 and an were classified as doubtfully progressive and No. l was called nonprogressive. Comparison of the lesions due to 5. 9.21.1.3. and those due to Group III woobacteria shows that the disease caused by Group III organisms tended to be self-liniting.‘ Calcification and fibrous encapsulation were minimal in the a. m; infected animals but were prominent in.the Group III infected calves. No Group III infected aninals died: both 5. m calves were killed in gm. The histological I'Flowever. subsequent unpublished data indicate that Group III nycobacteria of bovine origin can kill calves. 131 I .. . j. $‘[i"ll\f 1!.{1l I I. III I II II. I I ‘ ' rill- 'III II! II .II lit. I II I! 132 indications are that in.aany of the Group III infected calves the lesions may have become nonprogressive with the chance of sterilisation later. Even those animals with progressive lesions. such.as calf #5. had many encapsulated caseo-calcareous lesions which had already become nonprogressive. It is interesting to compare the pathological changes caused by the one culture when inoculated into more than.one calf. Thus. when 2.2 mg. of Culture 510-0 was inoculated into calf 2. generalised disease was produced. but some months later when 1 mg. of an inoculum from the same culture. which had been stored in a refrigerator. was injected into calf oz. only the regional lymph node was affected. The reisolant from calf 2 produced generalised disease when 1 mg. was injected into calf #3. Cul- ture 620-0 produced lesions only in the regional lymph nodes when 2.2 mg. were injected intradermally into calf 3. However. when a reisolant of this organism from calf 3 was injected at the dosage of 1 mg. intrader- mally into calf nu. generalised disease resulted. Culture 68C-0 produced generalised disease when 2.2 mg. were injected intradermally into calf h and 1 mg. of a reisolant of that organism produced generalised disease also in calf #5. Culture 1073-0 produced only an intradermal lesion at the site of inoculation in calf 13 but the reisolant from this calf pro- duced no microscopic lesions in calf_h0. ‘When 7lC-O was injected into calf. 7. it produced a primary complex. However. later. when the same organism was injected into calf 38. no microscopic lesions were seen. At the same time. a reisolant from calf 7 produced only a granuloma at the site of injection in calf 39. Thus. only two (107EbO and 788-0) of the 7 Group III cultures of bovine origin never produced significant lesions in calves. Thus. the Group III nycobacteria of bovine origin ranged from nonpathogenic to a pathogenicity approaching that of g. bovis. 133 .QZGID.III flgggbgcteriangf Swine Origin Seven of these cultures or their reisolants were injected into ll calves (Nos. 16. 23. 2h. 30. 31. 32. 33. 3b. 35. 36. and 37). Only one culture. 936-0. which was injected into only one calf (no. 16). produced any significant lesions. These were a caseo-calcareous granuloma 3 mm. in diameter in the left prescapular lymph node and a 35 x 15 x 10 mm. abscess in the dermis at the site of injection. Culture 172C1-l produced no microscopic lesions in calf 23 and only lesions at the intradermal inoculation sites in calves 30 and 31. Culture 193C2-l produced an intradermal inoculation site lesion only in calf 32. but in calf 33 no microscopio.lesion was detected. Culture l86C-l produced only intradermal inoculation site lesions in calves 3h and 36. Culture 16701-1 produced only an intradermal inoculation site lesion in calf 35 and no gross or microscopic lesions in calf 37. Thus. in this series. the Group III mycobacteria of swine origin had little pathogenioity for calves. m In W 2.: 22.-.9 29. $33.1; 9.12532 or the five calves (Nos. u6 through 50) momtud with 3 cultures of these organisms. only one (No. 47) had any lesions. This was a small granuloma at the site of injection and was the response to an inoculum of 10 mg. (approximately 109 to 101° organisms). Thus. the Group III mycobacteria of feed and soil origin used had no significant pathogenicity for calves. mm L__c°b‘°t°r1s After the inoculation of calves 11. 1a. and 17 had produced no evidence of pathogenicity. it was decided to inject four calves. Nos. .- 13“ 55-58. with larger doses. Two milligrams each were injected intrader- mally. subcutaneously. intramuscularly. and intraperitoneally. and 2 mg. were fed in a bolus of.the ration. All these calres were stressed acci- dentanyby exposure to an aerosol of ”-383. an orthopth phenol which is very irritant to the respiratory tract. All showed.symptoms of bronchitis and all had.a chronic bronchitis at necropsy. Nevertheless. calves 55-58 showed no evidence that these pseudochrome mycobacteria were pathogenic for them. Grann.I¥ulssahactasia. Because of the lack of pathogenicity. as in the ease of the pseudo- chromes. four calves (Nos. 51-5“) received 10 mg. of culture. and these calves were also unintentionally exposed to the lTb383 aerosol. Eleven calves were inoculated with live Group IV mycobacteria and one (No. 28) was inoculated with killed Culture ll7B-O. Only one calf (No. 51) showed any significant lesions. and these consisted of mostly nonprogressive encapsulated lesions in the regional lymph nodes. Calf 51 ‘was considerably debilitated due to the inhalation of an aerosol of 8T-383 and this had no doubt reduced his resistance to infection. Thus. it would.appear that some Group IVLmycobacteria can provoke primary com- plexes‘in calves. especially if the dosage is high and the animal is debilitated. However. even under such adverse conditions as these. calf 51 was able to render the infection generally nonprogressive and may have been able in time to have eliminated it completely. This ability of a Group IV’mycobacterium to form oaseo-calcareous granulomas is interesting in the light of the statement by Corpo.Runy°n tad Lester(l963) that ”present evidence suggests that the Group IV’mycobacteria do not produce the . characteristic features of caseating granulomatous disease." 135 W W Histologically. it was not possible to differentiate between lesions produced by g. 92:13 and those by atypical swoobacteria. In this series the lesions due to the atypicals were mostly nonprOgressive. and calci- fication. caseation and encapsulation were frequently seen. These features cannot be used to differentiate infection due to atypical mycobacteria from g. m infection - as similar changes may be seen in cattle infected. naturally with n. m. ' Acid-fast bacilli were not always detected by the new fuchsin- hematoxylin and eosin stain. even in advanced lesions such as those in calf #5. However. Braunstein and.Adriano (1961) have shown that fluores- cence microscopy can detect many more acid-fast bacilli in lesions than the standard Ziehl-Neelsen.method. Also. Hanks (1956) found that unless special precautions are taken. caseous material which frequently contains the mchbacteria can be lost.from the.section during processing. either during the preparation of the block or the staining of the section. Relationship Between Ulceration at the Skin Inoculation Site and Extent of Lesions These data are smarised in TABLES V through X. m 111 mm 91. m: exists At necropsy. twelve of fourteen calves had granulomas at the skin inoculation site and the remaining two had no observed lesions. Four of the twelve calves had no lesions other than at the skin inoculation site. The mere presence of a granuloma at the site of injection is not neces- sarily an indication of pathogenicity. since it is recognised that many foreign substances may stimulate a localized granulomatous response. However. ulceration at the injection site is a far better indication of 136 pathogenicity. For those calves on which data are available. of the six which had ulcers. three developed generalised disease. two had primary complexes and in one (calf 38) the skin ulcer healed completely. mmmumm Six of the eleven calves developed ulcers. but none developed even a primary complex. Calf 16. which did have a primary complex. did not develop an ulcer. MWW£MM§SEM One (calf #7) of these five calves developed an ulcer although none of them had primary complexes. However. calf #7 had been injected with 10 mg. of inoculum. WW Home of the seven calves developed skin ulcers or primary complexes. mums. Only one calf (Ho. 25) of the twelve developed an ulcer. but this had no primary complex. Calf 51. which did develop a primary complex from the intradermal injection. never had a skin ulcer. These results indicate that ulceration at the skin inoculation site was a good indica- tion of pathogenicity in the case of the Group III mycobacteria of bovine origin. feed and soil origin. pseudochrome mycobacteria and Group IV mycobacteria. However. ulceration due to swine origin Group III mycobac- teria did not correlate well with the pathogenicity. Six of these eleven calves had ulcers. but none of these had significant lesions: and the one 0‘11 fiith lesions (N0. 16) did.not have an ulcer at the inoculation site. The first six calves were inoculated with 0.1 mg. and 1.0 mg.. wet weight. of culture in separate locations. and both these doses were able 137 to induce ulcers if the swcobacterium was pathogenic. Host of the subse- quent cattle were injected with 1.0 mg. except where low or no pathogenicity was expected. and then the dose was sometimes increased to 10 mg. It must be aphasised that this is a small dose compared to those previously reported in the literatnre. Francis (1958) records the use of 50 mg. 31. m subcutaneously. 1+ mg. 1!. m intravenously. 50 mg. 31. M- m subcutaneously. 10-500 mg. 5. gig! subcutaneously. lO-ZOC mg. 3. £132 intravenously and 1 gm. )1. mm perorally. The fate of organisms injected intradermally. and the subsequent development of lesions in other organs is not simple. The lymphatic drainage from the hind leg is via the pepliteal lymph node to the super- ficial inguinal lnph nodes and thence to the deep. inguinals and internal iliacs. up the lumbar chain and finally into the thoracic duct. However. only one lymph node. the left prescapular. stands between the inoculation site on the lateral aspect of the left foreleg and the general circulation. After passing the left prescapular. acid-fast bacteria would be carried into the cervical duct. into the anterior vena cava and then immediately distributed to the lungs and the general circulation. It would some. then. that inoculation into the skin of the foreleg would give the organisms a better chance to cause a generalized infection. Intradermal inoculation also allows the organisms to lie in the animal body without the danger of immediate complete phagocytosis; thus. these organisms would be able to sensitise the animal. as has been postulated by Rich (1951). He believes that the necrosis and inflation which accmpam a tuberculous infection are caused by the bodys becoming sensitised to the tuberculo-protein. However. time is required to do this. art! if the organism is injected by routes other than the intradermal. they m be rapidly phagocytised and 138 broken down before sensitisation can take place. This is particularly likely to occur if they are in smallnumbera and/or of low virulence. Soltys and Jennings (1950) ' showed that after the subcutaneous injection of n. W in guinea pigs in doses of 0.3 mg. and 1.0 mg.. bacilli reach the lymph nodes within an hour. In those guinea pigs receiving the. smaller dose. the spleen was not positive for tubercle bacilli for 158-96 hours. However. tubercle bacilli were remvered from the spleens of the guinea pigs receiving the larger dose within an hour of injection. Bacilli were consistently in the blood only in the first few hours . No similar data for intracutaneous injection have been found. TABLE II shows that acid-fast bacilli were frequently recovered . from organs and lymph nodes. in which no lesions were detected either grossly or microscopically. It is possible that microscopic lesions were present in some of these lymph nodes. but doubtless a bacteremia was present in some animals. Horowits and Gorelick (1951) reconnend the bone marrow for the isolation and detection of tubercle bacilli in a bacillemia. but this did not give good results in our cases. Evaluation of Tuberculin Sensitivity in Relation to lesions Produced The Group IV. Pseudochromes. Swine Origin Group III and Feed and Soil Group III mycobacteria produced either no lesions or nonprogressive lesions which could resolve in man cases. It would seem desirable. therefore. to analyse the tuberculin test results to determine if an of these animals would have been removed by the caudal fold test. and whether the comparative cervical test would have helped to differentiate between those animals with progressive disease and those with either no lesions or only localised ones. These data'are sunarised in TABLES XI and III. 139 Interpretation of the tests was based on the 72 hour reading at the caudal fold site. The caparative cervical test results were computed from the 1&8 hour reading. An increase in skin thickness at the site of the mammalian injection which was greater than the increase at the avian site by‘ 5 n. was called a positive. 9cm Ill We 93. mm: m Group III wcobacteria of bovine origin were by far the most pathogenic for cattle of the atypical ”cobacteria. Of the ten animals infected with these organisms on Idtich comparative cervical tuberculin tests were conducted. there were three generalised cases (Ice. 1&3. M. 1+5). two cases (Hos. 7. 1&2) with lesions in the regional lymph nodes. three cases (Nos. 10. 13. 39) with lesions at the skin inoculation site only and two cases (Nos. 38. ‘50) with no gross or microscopic lesions. All three generalised cases gave positive caudal fold tests (the minimum increase being 15 Is.) and positive comparative cervical tests. Calves 7 and 1&2 with only primary complexes gave different responses. Calf 7 had a 6 mm. caudal fold increase at 72 hours. but at #8 hours the avian increase was 10 -. greater than that at the manalian site. Thus. the two tests gave conflicting results. vis.. a positive caudal fold and a negative comparative cervical test. Calf #2 had only a 1.5 u. increase at the caudal fold site at 72 hours. The avian response was 9 n. greater than the mammalian at 1&8 hours in the cervical test and thus negative. The remaining five calves all had negative caudal fold and cervical tests. although two of them (Nos. 10 and 39) had 3 ms. and 2 mm. increases. respectively. in the caudal folds. In no case would the use of the comparative cervical test have removed nonprogressive cases from the herd. The caudal fold test would [I'l‘lllI‘ll‘IllI ‘1' i {III-I. |Il ll'll‘ II..- 1"- Iv- I III-It‘ll! I ‘II? in“ t 1&0 have undoubtedly caused.the removal of calf 7. If it is felt that the tuberculin test should.remove all cattle with lesions. then the compara- tive cervical test failed to do this in two cases and the caudal fold test chose one of these and left the other. mmmmmmm Only one animal (Ho. 16) had lesions. which consisted of an abscess in the skin at the inoculation site and a granuloma in the left prescapu- lar lymph node. The caudal fold test of Ho. 16 was negative. and the cervical test showed a.maximal sensitivity to avian tuberculin. Thus. if the comparative cervical test were applied with the mammalian response exceeding the avian by at least 5 mm. before the animal was called posi- tive. this animal.would have been declared negative. On pathological grounds. it is possible that the infection could have been overcome and even eliminated. As isolations of'mycobacteria were made only from the left prescapular lymph node and the skin inoculation site. this would tend to support the theory of only localised infection. Of the remaining nine animals tested. only three gave caudal fold test increases greater than 3 mm. 72 hours after injection. However. on the comparative cervical test. nine calves had maximal reaction to avian tuberculin. and only one (no. 23) had a.meximal response to mammalian tuberculin. This calf 23 had no gross or microscopic lesions and. as the mammalian tuberculin response‘was only 2 mm. greater than that of avian tuberculin. it would have been declared negative by the comparative test and by the criteria set up. Thus. in this group of calves. the caudal fold test would have resulted in the condemnation of three animals without lesions and failed to identify one (Ho. 16) with localised lesions. The comparative cervical test would not have designated any animals as positive. 141 can In W 21. mm and mu 9mm None of the five animals infected with a-oup III sycobacteria of feed or soil origin had lesions. and none was positive to the caudal fold test. The largest increase was 3 an. in the case of calf 50. In all cases the comparative cervical test showed greater sensitivity to avian tuberculin. and thus. all calves were considered negative. mm mm None of these seven calves had significant lesions. but one (calf 55) was positive to the caudal fold test with a 6 an. increase. However. it and all others gave negative comparative cervical tests. 2232 I! mama Chily one (No. 51) of the eleven animals had significant lesions. and these were mostly encapsulated nonprogressive lesions in the regional lymph nodes. This animal. which had received the large dose of 10 mg.. had an increase in its caudal fold of 3.5 -.. but the comparative cervi- cal test was negative. The only other significant increase in the caudal fold was in calf 9. which had a 7—n. increase. although it had neither gross‘nor microscopic lesions. In all cases the comparative cervical tests were negative. 142 Eyeluation of Tuberculin Sensitivity Produced by Killed Wcobacteria TABLE XIII summarises the data on the results of the tuberculin tests on calves inoculated with killed mycobacteria. All calves gave negative tests to the comparative cervical tests. but calf 26. injected with killed I!- ngig. had a 5 an. increase in the thickness of its caudal fold at 1&8 hours. Calves 27 and 28 had small increases in the thickness of their caudal folds. but calf 29. injected with killed Group III myco- bacteria. had no response. These findings indicate that killed mycobacteria can induce sensitivity but the comparative cervical test showed a greater sensitivity to avian tuberculin rather than mammalian. VI. SUMMARY Fifty-five calves of various breeds and both sexes. and between six and ten months of age. were inoculated with swcobacteria. Each calf received a single culture. Unless otherwise specified. calves were inocu- lated with either 1 mg. or 2.2 mg.. wet weight. of organisms intradennally on the legs. They were tuberculin-tested on the caudal fold with mannalian tuberculin. by the comparative cervical method using avian and mammalian tuberculins and johnin. and examined by necropsy 8-12 weeks after inoculation. ‘ Thirty-seven calves were inoculated with Mon Group III mycobacteria. 11 with Group IV wcobacteria. 2 with g. m. l with 35. 5141-12 and 1 each with killed cultures of 14. m, l!- m. a Group III of bovine origin and a Group IV mycobacterium. Four of seven Group III cultures of bovine origin. inoculated into 14 calves. produced either a primary complex or generalised disease in 8 calves. ‘hlo other cultures failed to produce a primary complex and another produced this in only one of three calves. The six different Group III cultures of swine origin were all isolated from swine mesenteric lymph nodes and were inoculated into 11 calves. Only one culture produced a primary complex with granulomas at the inoculation site and in the left prescapular lymph node. Of seven calves inoculated with a total of four cultures of 'pseudo- chromes'. five had no lesions and two had only small granulomas at the inoculation sites. Hour of these calves had each received a total of 10 mg.. wet weight. of organisms. 2 mg. being administered by each of the 1&3 1M peroral. subcutaneous. intradermal. intramuscular and intraperitoneal routes. Five calves inoculated with. three cultures of Group III mycobactoria of soil or cattle feed origin did not develop primary complexes. Six cultures of Group IV awcobacteria were injected into 11 calves. 1+ of finish received a total of 10 mg.. administered by the peroral. subcu- taneous. intradermal. intraperitoneal and intramuscular routes. Four of these cultures were isolated from bovine "skin lesions“. Two of them caused no lesions in the experimental calves. Q). culture produced a small intradermal granuloma in one calf. but no lesions in a second calf receiving the 10 mg. dose. The fourth culture produced no lesions in one calf. but the calf which received 10 mg.. had encapsulated nonprogressive granulomas at the skin injection site and in the lymph nodes draining the intradermal. subcutaneous and intramuscular injection sites. Both calves infected with 5. m developed generalised disease. The calf injected with M. avium developed only a module at the injection site. Home of the calves injected with killed cultures developed lesions. In every case the comparative cervical test differentiated between those calves with progressive disease. as determined by histological exami- nation. and animals with either no lesions or localised nonprogressive lesions. Four animals inoculated with live qcobmteria and with no lesions. and one. with a nonprogressive lesion. gave positive caudal fold tests. in calves inoculated with killed. wcobacteria gave negative comparative cervi- i cal results. but one gave a positive caudal fold reaction. LISTWWCES Alvarez and Tavol. 1885. Recherches sur le bacille do lustgarten. . Arch. MeioInPath" 6:303. Cited by Xalabardor. 1961. Anowmous. 1962. Tuberculosis procedures manual. Mich. Dept. Agric.. Livestock Disease Control Division. and U. 8. Dept. Agric.. Animal Disease Eradication Division. Lewis Cass Building. lensing. Hichigan. Baum. H. 1912. Das Lymphgofasssystem dos Rindos. A. Hioschwald. Berlin. Benjamin. M. H. 1961. Outline of veterinary clinical pathology. Iowa State Univ. Press. Ames. Iowa. Boddie. G. I. 1962. Diagnouic methods in veterinary medicine. 5th ed. Lippincott. lhiladelphia. Pa. :289. Braunstein. H. and Adriano. S. H. 1961. Fluorescent stain for tubercle bacilli in histologic sections. I. 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