TUBERCULOPOLYSACCHARIDE AND TUBERCLELC‘PHOSPHATEDE PASSEVE AGGLUTENATEON \‘Q‘H‘H BOWNE AND PQR‘CENE gER‘QMS’ This}: for “are Dogma of M. 5. MICHIGAN STATE UNIVERSITY Patsy June Robinson 1964 I THESIS LIBRARY Michigan State University ABSTRACT Robinson, Patsy J. (Michigan State University, East Lan- sing, Michigan). Tuberculopolysaccharide and tuberculophos- phatide passive agglutination with bovine and porcine serums. 1964.--Tuberculopolysaccharide and phosphatide specific antibodies were measured by the hemagglutination (HA) and kaolin-phosphatide (KP) tests, respectively. The serums- tested were from calves and swine inoculated with mycobac- teria and from tuberculin positive cows from gross-lesion and no~gross-lesion herds. Serum titers were 20 or less from all except four of 120 animals prior to experimental infection, and were considered negative. Two cows from a gross-lesion herd had fourfold (anamnestic—like) response in HA and KP titers 3 to 13 days after tuberculin testing and progressive disease at necropsy. Two cows from a no- gross—lesion herd did not respond anamnestically to tuber- culin and had no lesions at necropsy. Animals inoculated with pseudochromes, Group IV and Group III organisms (inanid mate origin) had few or no lesions and low serum titers. Group III organisms (porcine origin) caused few lesions in calves but the calves responded anamnestically to tuberculin. Swine inoculated with Group III organisms (porcine origin) had fourfold increases in HA and KP serum titers and progres- sive disease at necropsy. Cattle's serologic responses were altered quantitatively by the route of administration of Group III organisms (bovine origin), intradermally, intra- uterine, or orally. Specific phosphatide extracts prepared from various mycobacteria did not increase the specificity or diagnostic value of the test. TUBERCULOPOLYSACCHARIDE AND TUBERCULOPHOSPHATIDE PASSIVE AGGLUTINATION WITH BOVINE AND PORCINE SERUMS BY Patsy June Robinson A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1964 ACKNOWLEDGMENTS The author wishes to express her sincere thanks to Dr. V. H. Mallmann and Dr. w. L. Mallmann for their guidance during the progress of the research and prepara- tion of the manuscript. Thanks are also extended to Dr. J. A. Ray, Dr. M. D. McGavin, Dr. L. Goyings and the other members of the Tuberculosis Research Project for their assistance and cooperation. TABLE OF CONTENTS Page INTRODUCTION. . . . . . . . . . . . . . . . . . . . . l HISTORICAL REVIEW . . . . . . . .4. . . . . . . . . . 3 Agglutination . . . . . . . . . . . . . . . . . . 3 Hemagglutination. . . . . . . . . . . . . . . . . 4 Hemagglutination in study of tuberculosis . . . . 7 Kaolin agglutination. . . . . . . . . . . . . . . 16 Tuberculopolysaccharides. . . . . . . . . . . . . 16 Tuberculophosphatides . . . . . . . . . . . . . . l9 Atypical mycobacteria . . . . . . . . . . . . . . 22 MATERIALS AND METHODS . . . . . . . . . . . . . . . . 25 Cultures for inoculation. . . . . . . . . . . . . 25 Preparation of cultures for inoculation . . . . . 25 Experimental animals. . . . . . . . . . . . . . . 28 Collection of serum . . . . . . . . . . . . . . . 34- Hemagglutination (HA) reagents and test . . . . . 34 Collection and sensitization of sheep erythrocytes. . . . . . . . . . . . . . . . . 34 Inactivation and adsorption of serums . . . . 35 Serum dilutions . . . . . . . . . . . . . . . 36 Hemagglutination (HA) test. . . . . . . . . . 36 Kaolin-phosphatide (KP) reagents and test . . . . 37 TME buffer solution . . . . . . . . . . . . . 37 Preparation of TME buffered saline. . . . . . 37 Preparation of phosphatide-methanol antigen Preparation and sensitization of kaolin . . . Inactivation and dilution of serums . . . . . Kaolin-phosphatide (KP) agglutination test. . . . Preparation of experimental phosphatide extracts. RESULTS . . . . . . . . . . . . . . . . . . . . . . . Preliminary determinations of the HA test . . . . Serum HA and KP titers of uninoculated animals. . Calves 1 through 25 (Lot I) . . . . . . . . . . . Calves inoculated with Mycobacterium bgzis. . Calves inoculated with Mycobacterium 23323, . Calves inoculated with Group III (bovine origin) mycobacteria. . . . . . . . . . . . . Calves inoculated with Group III (porcine origin) mycobacteria. . . . . . . . . . . . . Calves inoculated with pseudochrome myco- baCteriao O O 0 0 0 O 0 O 0 0 0 O O o o O O O Calves inoculated with Group IV mycobacteria. AdUlt COWS (LOt II) 0 0 0 O O O o 0 O O O O O 0 0 SWine (LOtS III and IV) 0 O 0 O O 0 0 0 O O 0 O O Swine inoculated intradermally with M co- bacterium bovis . . . . . . . . . . . . . . . Swine fed Mycobacterium bovis . . . . . . . . Swine inoculated intradermally with M co- bacterium avium . . . . . . . . . . . . . . . Swine inoculated intradermally with Group III (porcine origin) mycobacteria . . . . . . . . Swine fed Group III (porcine origin) mycom bacteria. . . . . . . . . . . . . . . . . . . Swine inoculated intradermally with Group III (inanimate origin) mycobacteria . . . . . . . Page 38 39 40 40 40 43 43 43 43 44 51 51 52 53 54 54 54 64 64 64 65 65 66 Uninoculated swine. . . . . . . . . . . . Calves 26 through 58 (Lot V). . . . . . . . . Calves inoculated with heat-killed myco— bac teria O O O O O O O O O O O O O O 0 O O Calves inoculated with Group III (bovine origin) mycobacteria. . . . . . . . . . . Calves inoculated with Group III (porcine origin) mycobacteria. . . . . . . . . . . Calves inoculated with Group III (inanimate origin) mycobacteria. . . . . . . . . . . Calves inoculated with pseudochrome myco- bacteria. . . . . . . . . . . . . . . . . Calves inoculated with Group IV mycobacteria. Calves inoculated intradermally (Lot VI). . . Calves inoculated with SlC-O. . . . . . . Calves inoculated with 68C-0. . . . . . . Calves inoculated with SOB-O. . . . . . . Heifers inoculated intrauterine (Lot VII) . . Heifers inoculated with SlC—O . . . . . . Heifers inoculated with 68C-0 . . . . . . Heifers inoculated with SOB-O . . . . . . Calves of aerosol exposure (Lot VIII) . . . . Calves inoculated with SlC-O. . . . . . . Calves inoculated with 68C-0. . . . . . . Calves inoculated with SOB-0. . . . . . . Kaolin-phosphatide serum titers of calves and swine inoculated with mycobacteria using phos- phatides extracted from various mycobacteria. Kaolin-phosphatide serum titers of calves 30 through 50 inoculated with mycobacteria . . . Page 66 66 7O 70 71 71 72 72 72 73 73 73 77 77 77 78 78 78 78 79 79 84 Page DISCUSSIONO O O O O O O 0 0 O 0 O O O O O O 0 O 0 O C 89 SUMMAR.Y O 0 O 0 O 0 O 0 O O O 0 0 0 O O O O 0 O O O O 106 LITERATURE CITED 0 0 O 0 0 O O O 0 O O O 0 O O 0 0 O O 109 Table 1. LIST OF TABLES Page List of animals inoculated with mycobacteria. . 26 Serial tuberculopolysaccharide specific hemag- glutination (HA) serum titers of calves (1-25) inoculated with mycobacteria. . . . . . . . . . 45 Hemagglutination (HA) and kaolin-phosphatide (KP) serum titers and pathology reports of . calves (1-25) inoculated with mycobacteria. . . 49 10. ll. Hemagglutination (HA) and kaolin-phosphatide (KP) serum titers and pathology reports of four cows before and after tuberculin testing. . . . 55 Tuberculopolysaccharide specific hemagglutina- tion (HA) serum titers of swine inoculated with mycobacteria . . . . . . . . . . . . . . . S7 Hemagglutination (HA) and kaolin-phosphatide (KP) serum titers and pathology reports of swine inoculated with mycobacteria. ... . . . . 61 Hemagglutination (HA) and kaolin-phosphatide (KP) serum titers and pathology reports of calves (26—58) inoculated with mycobacteria . . 67 Hemagglutination (HA) serum titers and pathol- ogy reports of calves inoculated intradermally (ID), intrauterine (IU), or exposed to an aerosol (AE) of Group III mycobacteria of bovine origin . . . . . . . . . . . . . . . . . 74 Comparison of kaolin-phosphatide (KP) serum titers using Takahashi's reagents and prepared reagents 0 O o o O O 0 O O 0 O O O O 0 0 O O O 0 80 Kaolin~phosphatide (KP) serum titers of calves and swine inoculated with mycobacteria using phosphatide extracts from various mycobacteria. 81 Kaolin-phosphatide (KP) serum titers of calves inoculated with mycobacteria using phosphatide extracts from various mycobacteria. . . . . . . 85 INTRODUCTION The need exists to differentiate between tuberculous infection and disease, and between active and closed cases. Since the demonstration of the etiologic agent of tubercu— losis, the need of a single diagnostic test has been recog- nized and investigated. The tuberculin test is one of the most widely used tests for the detection of tuberculosis but it does not distinguish between past and present infec- tion or disease. Polysaccharide specific, protein specific and phos- phatide specific antibodies may be present in the serums of infected individuals and animals. Most of the serologic studies have been made with serums from human cases of tu- berculosis. As yet, no one test or tests have proved to be reliably diagnostic or differential. As the incidence of bovine tuberculosis has been reduced, the problem of the tuberculin positive animal which has no gross lesions at slaughter has become more apparent and acute. Mycobacteriumlbgzi§_has been isolated from some of the cattle. From far more, mycobacteria have been iso- lated which are not the classical pathogens, and have been Called atypical, anonymous or unclassified mycobacteria for want of a better name. Their role in the tuberculosis <1r tuberculosis-like disease and in inducing tuberculin sensitivity is presently under investigation. In conjunc- tion with those investigations, studies were made of the serums from the calves and swine inoculated with the myco- bacteria. Polysaccharide specific and phosphatide specific antibodies in serums have been measured by hemagglutination and kaolin particle agglutination. This is a report of the serologic studies. HISTORICAL REVIEW Agglutination. Agglutination, a visible reaction which may be observed when a particulate antigen is mixed with serum containing specific antibodies, was reported in 1896 by Gruber and Durham. Since that time applications of the phenomenon have been the most useful serologic technique for the diagnosis of infectious diseases. Unknown organisms or antigens can be identified by the reaction with specific serums. The presence and relative concentrations of antibodies can be determined by using known antigens. The diagnostic significance of the latter must be determined empirically. Antibodies may occur in serums of healthy individuals not known to have had previous contact with the antigens. Agglutination tests may be performed on surfaces such as glass slides, in containers such as test tubes, or the reaction may be measured photometrically. Of the three the surface method is the simplest and quickest to perform, is less sensitive, and is generally used qualita— tively. The test tube method is used quantitatively by adding antigen to decreasing amounts of serum or serum to decreasing amounts of antigen. The titer is recorded as the reciprocal of serum or antigen which caused detectable agglutination . ti. an ti C0 by De th of it Photometric determination was introduced by Hirst and Pickels (1942) with agglutination of erythrocytes by influenza virus. The virus agglutinates the erythrocytes, and the presence of antibodies in a serum is detected by the specific inhibition of agglutination. Photometric de- terminations are made after the mixtures begin to settle and three layers are present in the test tube: the top clear layer devoid of erythrocytes, the middle layer of sedimenting cells of uniform density, and the bottom layer of cells which settled more rapidly. The optical density of the middle layer is measured and compared with a control tube. Initially, suspensions of bacterial cells served as the particulate antigen. Subsequently extracted bacterial antigens have been adsorbed onto the surface of various par- ticles such as erythrocytes (Middlebrook and Dubos, 1948), collodion (Jones, 1927), carbon (Meyer and Pic, 1936), latex (Singer and Plotz, l956a,b), and kaolin (Takahashi and Adachi, 1960). These coated particles may be agglutinated by specific antibodies. Since the bacterial cells are com- posed of many antigens, exclusion of those antigens other than the one which is species or strain specific, generally yields a more specific test. Hemagglutination. Hemagglutination, the agglutination of erythrocytes, was first noted in the 19th century when it was observed that transfusions to human beings of blood 0:] (ii in he an de; he from sheep or other domestic animals resulted in fatal re— actions. Landois (1875) found that if human blood was mixed in xitrg with blood from other animals, the blood cells were lysed or agglutinated. The studies by Landsteiner and others on the blood cell groups of man followed. In 1900 Landsteiner discovered that the blood of human beings could be divided into three distinct groups, A, B, and O, on the basis of hemagglutination reactions. The fourth and rarest group,- AB, was discovered in 1902 by von Castello and Sturli. Through extensions of those studies, the complexities, presence of isoantibodies, phylogenetic relations, and many interesting phenomena concerning the blood cells of humans and higher animals have been established (Wiener, 1939). As a result of the above and other studies, hemag- glutination was investigated for use with other antigen- antibody systems. Davidsohn (1927, 1929, 1930, and 1933) demonstrated that sheep erythrocytes were agglutinated by heterophilic agglutinins in serums from individuals who had received injections of materials containing horse serum protein. Paul and Bunnell (1932) employed the Davidsohn technique to determine the increased amounts of heterophilic agglutinins in patients with infectious mononucleosis. A test was developed by Davidsohn (1937) which is now used routinely in the diagnosis of this disease. Another application of hemagglutination measures "cold agglutinins," so called because their action occurs only at low temperatures and is reversible when warmed to 37 C (Turner, 1943). Cold agglutinins are found in patients with atypical or virus pneumonia. Hemagglutination by virus was discovered in 1941. Allantoic fluid from chicken embryos infected with influenza virus agglutinated the embryonic chicken blood. The agglu~ tination was due to the direct action of virus particles on the erythrocytes and required no specific antibodies (Hirst, 1941; McClelland and Hare, 1941). Neter (1956) described eight types of microbial hemagglutination: a) the direct microbial hemagglutination reaction. Kraus and Ludwig (1902) observed clumping of erythrocytes in the presence of staphylococci and vibrios. (Later work has shown that many different bacteria possess the ability to agglu- tinate erythrocytes). b) the indirect, passive, or conditioned hemagglu- tination reaction. Keogh, North and Warburton (1947) observed that polysaccharide antigen would adsorb onto the surface of erythrocytes which subsequently was agglutinated by specific anti- body added to the mixture. Boyden and Suter (1952) have suggested the term "hemosensitin" for those antigens which adsorb spontaneously onto erythrocytes and render them sensitive to agglutination or lysis by antibody against the adsorbed antigen. The term would exclude and distinguish from those substances which agglu- tinate erythrocytes directly such as certain hemagglutinating viruses. c) bacterial hemagglutination of erythrocytes treated with antigenaantibody mixture. Boyden and Anderson (1955) found that untreated erythrocytes exposed to a mixture of tuberculoprotein and homologous antibody resulted in hemagglutination. d) bacterial hemagglutination of erythrocytes pre- treated with tannic acid. Erythrocytes which had been previously treated with tannic acid and the microbial antigen, were agglutinated by specific antibody (Boyden, 1951). e) bacterial hemagglutination by antibodies to pro- tein antigens which have been attached to erythro- cytes by chemical linkages. Pressman, Campbell, and Pauling (1942) devised such a method employ- ing bis-diazotized benzidine as the protein con- jugating material. Cole and Farrell (1955) suc- ceeded in attaching to formalinized erythrocytes tuberculin PPD via tetrazotized benzidine. f) erythrocyte linked antigen hemagglutination test. Non-bacterial protein antigens are attached to incomplete Rh (Coombs, Howard and Wild, 1952), ox red cell (Coombs, Howard and Mynors, 1953), and Forssman antibodies (Coombs and Fiset, 1954). The erythrocyte antibodies then act as Schleppers (carriers) for the protein and when treated with an antigen become agglutinable in the presence of homologous antibody. 9) bacterial panagglutination or the Thomsen- Friedenreich phenomenon. This was first observed by Thomsen in 1927 and extensively studied by Friedenreich in 1930. Enzymatic activity of certain bacteria uncovers a substrate referred to as T receptor or antigen, which reacts with the corresponding T antibody. h) bacteriogenic hemagglutination. In 1940 Davidsohn and Toharsky reported that certain bacteria pro- duce changes in normal serum which result in a panagglutinating capacity. Hemagglutination in the study of tuberculosis. Middle- brook and Dubos (1948) used the principle of hemagglutina- tion demonstrated by Keogh, North, and Warburton (1947, 1948) in the study of experimental tuberculosis. Incuba- tion of sheep erythrocytes with culture filtrates of tubercle bacilli sensitized the sheep erythrocytes to agglutination by the serums of tuberculous human beings and experimentally infected laboratory animals. Methanol extracts of phenol- acetone treated tubercle bacilli adsorbed onto the surface of sheep erythrocytes which were then agglutinated by serums from tuberculous patients and rabbits experimentally infected with an attenuated strain of‘M.‘§g1£§, the Bacillus of Cal- mette and Guerin (BCG). The hemagglutination reaction could be inhibited by extracts and fractions of tubercle bacilli added to the serum before the introduction of the sensitized erythrocytes into the system (Middlebrook, 1950a). Fisher and Keogh (1950) modified this system by adding a small amount of complement. Hemolysis, rather than agglutination, occurred. Modifications of the original Middlebrook-Dubos he- magglutination test have been developed in an attempt to make the test easier, faster to perform, less expensive and more specific. A practical modification of the test was developed which appeared to be equally specific for antibodies produced by the tubercle bacillus. Old Tuber- culin (Lederle) four times the standard strength, diluted 1 to 15 in buffered isotonic saline, was substituted for the extract of the tubercle bacillus (Scott and Smith, 1950). Erythrocytes from species other than the sheep have been used in hemagglutination tests. Gernez~Rieux and Tacquet (1952) compared erythrocytes from sheep, human Group "0," rabbits, guinea pigs, horses, cattle and chick- ens. Variation in the intensity of agglutination was observed W1 an (Ti? .r—\ ’..._a \f‘) “RP: ~¢!\Ju Li ti. mq :1 ‘ (I) < 1‘85 with erythrocytes from these different species. Human eryth- rocytes agglutinated less strongly than sheep erythrocytes. Sohier (1952) and Adcock, et al. (1951) compared sheep eryth- rocytes and human Group "0" erythrocytes and found the human erythrocytes to be effective. In addition, the natural antisheep agglutinins did not have to be adsorbed from the human serum. A ”drop technique" was developed by Rheins, et a1. (1954). Drops of the serum and reagents were used instead of the exact amounts delivered from a pipette. A slide modification was devised by Thalhimer and Rowe (1951) which utilized glass slides rather than test tubes. Both tech- niques proved to be as sensitive as the original test and have the added advantages that small amounts of serum and reagents are required, and the incubation period was short- ened to three minutes in the latter test. During the years 1950 to 1954 many investigators evaluated the hemagglutination test by clinical and experi- mental observations (Adcock, et al., 1951; Anderson and Platou, 1951; Fleming, et al., 1951; Hall and Manion, 1951; Kirby, et al., 1951; Maillard and Gagliardo, 1951; Rothbard, 1951; Hentel and Guilbert, 1952; Maillard, 1952; Mollov and Kott, 1952; Schwartz, et al., 1952; Spain, et al., 1952; Hollander, et al., 1953; Rabe and Spicer, 1953; Choremis, et al., 1954). According to some of these investigators, the test was of diagnostic value. Others reported it to 10 be of little or no value. The serums used in the studies were primarily from hospital patients, patients with active tuberculosis and non-tuberculous patients with miscellaneous diseases or from normal individuals before and after vac- cination with BCG. Fleming, et a1. (1951) studied serums from over 400 individuals. Patients with active tubercu- losis had the highest titers, and nonutuberculous patients and normal individuals usually had no or low titers. Titers decreased when the patient was improving or in the terminal stages of the disease (Anderson and Platou, 1951; Babe and Spicer, 1953). Individuals who were tuberculin negative after repeated tuberculin tests usually had positive hemag- glutination titers after vaccination with BCG (Smith and Scott, 1950; Haley, et al., 1952). What constitutes a significant titer is controver- sial. Middlebrook (1950b) reported that a positive titer of 8 or higher was presumptive evidence of disease due to tubercle bacilli or to a mycobacterium very closely related in antigenic structure. Kirby, et al.,(1951) reported that a large percentage of normal individuals had positive titers of 8 or higher, and for that reason, the test was not re- liably diagnostic. Rabe and Spicer (1953) reported the test as being acceptably specific on the basis of a "sig- nificantly elevated" titer of 64. With the latter criterion, the test was positive with serum from 90% of thirty tuber- culous patients. A titer of 32 ("doubtful significance") 11 was present in 87% of the tuberculous patients and 4% of the non-tuberculous patients. Studies have been made of the serums from cattle. The percentage of positive reactions in tuberculous cattle was higher in those animals suffering from visceral tuber- culosis than in those animals with isolated lymphatic le- sions. Positive reactions were obtained in 85 to 90% of cattle with active visceral tuberculosis (Sohier, et al., 1950; Fisher and Gregory, 1951; Gernez-Rieux and Tacquet, 1952). Richey, Mack, and Stafseth (1954) studied the he- magglutination reaction of chickens experimentally infected with avian tubercle bacilli. Adult chickens which had pos- itive avian tuberculin tests all had antibody titers of 32 or higher. Of 41 chiCkens hatched from eggs inoculated with tubercle bacilli, seven reacted to tuberculin at seven months of age and had lesions when sacrificed at eight months of age. The seven had hemagglutination titers of 16 or higher. The remaining 34 chickens varied in response to tuberculin, none showed lesions, and 91% had hemagglu- tination titers of 4 or higher. Hemagglutination tests performed before and after tuberculin testing on serums from normal adult tuberculin negative chickens showed no increase in hemagglutination titer. Boyden (1951) treated sheep erythrocytes with tan- nic acid. These cells then preferentially adsorbed 12 tuberculoprotein and were agglutinated by specific antibodies. Agglutination could be inhibited by adding culture extracts to the system before the introduction of sensitized eryth- rocytes. Takahashi, et al. (1961a,b) used the hemagglutina- tion test to measure three different antibodies, the tuber- cu10protein, the tuberculopolysaccharide, and the tuberculo- phosphatide specific antibodies in serums from patients with tuberculosis and experimental aniamls. Experimental animals were inoculated with M, bgyi§_(BCG) and a virulent M. tuberculosis (strain Nakano). Polysaccharide specific and protein specific antibodies were detectable regardless of the route of inoculation or the virulence of the bacilli. The phosphatide specific antibody was elicited under con- ditions in which the authors believed there had been.i£;yixg multiplication and destruction of the bacilli. The pres- ence of phosphatide antibody reportedly was indicative of progressive infection. Takahashi, et al., (1961b) studied the three anti- bodies with serums from tuberculin positive patients with active tuberculosis and tuberculin positive healthy indi- viduals. The polysaccharide specific antibody was present in all individuals of both groups. Approximately 54% of the tests for the protein specific antibody were positive and 46% were negative. The phosphatide specific antibody was found only in those individuals with active tuberculosis, 13 but not detectable in 20%. In the healthy tuberculin posi- tive individuals, all had polysaccharide specific antibodies, 44% had protein specific antibodies, and none had phosphatide specific antibodies. Results of tuberculin skin tests, ex- tent of tuberculous lesions, types of disease, existence of cavities, sputum findings and disease activity, and anti— body titers were compared. There was no relationship between the titers of the three types of antibodies and tuberculin skin hypersensitivity. In all other instances there was a direct correlation between the phosphatide specific antibody titers and disease. Although the hemagglutination test and its various modifications are controversial as to their use as aids in the diagnosis of tuberculosis, it has been found useful for other purposes. Investigators used the hemagglutination procedure to study the antigens of the tubercle bacilli (FiSher, 1951; Meynell, 1954; Rheins and Thurston, 1955; Thurston and Rheins, l956a,b,c,d). Meynell (1954) demonstrated that the polysaccharide and protein antigens present in a protein precipitate were composed of 88% protein, 5% polysaccharide and 7% nucleic acid. They could be separated by adsorbing the polysaccharide antigen on normal sheep erythrocytes and the protein antigen on tanned sheep erythrocytes. Although polysaccharide anti- gen would adsorb onto tanned cells, the smaller concentra— tion of protein necessary for adsorption allowed dilution 14 of the suspension and the polysaccharide portion adsorbed onto the tanned cells was negligible. Sensitization of normal erythrocytes with only polysaccharide was possible with concentrated solutions because protein does not report- edly adsorb onto normal cells under any conditions. Tanned cells are sensitized by protein only by using antigen solu- tion so dilute that the concentration of polysaccharide is too small to produce detectable sensitization. Polysaccharide and phoSphatide antigens are found on the surface of the bacterial cell while the protein anti- gen is a "deep" antigen. Bacillary suspensions combine with the polysaccharide and phosphatide specific antibodies. They do not combine with the protein specific antibody. The protein specific antibody remains in the supernatant (Meynell, 1954). Successive adsorption of Old Tuberculin (Lederle) with sheep erythrocytes permitted demonstration of anti- bodies which probably are directed against antigens not detected by the usual sensitizing procedures (Rheins and Thurston, 1955). In a series of experiments Thurston and Rheins (l956a,b,c,d) studied extract antigens prepared from M. 332133 (BCG), _I‘_'I_. hlei, and M. tuberculosis (strain H37Rv). Water and saline extracts of the organisms, as well as the extracts prepared by the older, more tedious methods and Old Tuberculin, sensitized sheep erythrocytes to agglutina- tion by specific antibodies. Water and saline extract-treated 15 sheep erythrocytes exhibited serologic specificities not seen with Old Tuberculin-sensitized erythrocytes. Antigens from aqueous extracts of fl?.§2!$§,(BCG) and in Old Tuberculin were adsorbed to different sites on the erythrocytes. Tan- ning of erythrocytes did not result in demonstration of antibodies other than that observed with normal erythrocytes. Lipids and lipid extracts such as lecithin and ceph- alin inhibit sensitization of erythrocytes with polysacchar- ide (Boyden and Grabar, 1954). The polysaccharide is believed to attach to a phospholipid on the erythrocyte surface. Gerstl, et al., (1955) detected antibody activity in apparently negative serums from tuberculous patients after fractionation of the serums. Serums which were nega- tive by the polysaccharide test caused hemagglutination and complementmfixation after fractionation by the cold ethanol method of Nichols and Deutsch (1948). The fractions were labeled supernatant A, largely albumin; precipitate B, largely beta and gamma1 globulins; precipitate C2, gamma2 globulins. Serums from eight individuals had hemagglutinins in the C2 precipitate. Seven of eight serums had complement fixing antibodies in the B precipitate. Serums from non— tuberculous patients did not react in either test before or after fractionation. Injections of Old Tuberculin into guinea pigs which had previously received tubercle bacilli resulted in the appearance of increased amounts of polysaccharide antibody 16 in the serum (Boyden and Suter, 1952). The increase appeared within four to seven days with a maximum increase at ten days. By 28 days the increase in antibodies had disappeared in most of the animals. There was no relationship between the amount of skin hypersensitivity and the antibody titers. The amount of old Tuberculin did not significantly change the antibody response. The route of injection of Old Tuberculin made no difference in antibody response. Kaolin agglutination. Takahashi and Hukae (1960) substi— tuted kaolin for sheep erythrocytes as the antigen carrier in the passive hemagglutination test. They compared the hemagglutination and the kaolin agglutination tests using phosphatide antigens prepared from M. tuberculosis (strains Nakano, H37Rv, and Aoyama B), and.§.‘bgyi§,(BCG) and serums from patients with pulmonary tuberculosis. The kaolin ag- glutination test was twofold to fourfold times as sensitive as the hemagglutination test. Prior to this time kaolin had not been used as an antigen carrier in agglutination tests. Tubercu10polysaccharides. The polysaccharides of the tubercle bacilli have been studied extensively since Koch first examined extracts of cells and culture filtrates in 1882. They have been primarily of three sources, those associated with the somatic portion of the cell (Chargaff and Schaefer, 1936; Menzel and Heidelberger, 1939; Tennent and Watson, 1942; Siebert, 1950); those associated with the 17 lipids (Anderson, Lothrop and Creighton, 1930); and those present in culture filtrates (Anderson, Peck, and Creighton, 1940; SutomNagy and Anderson, 1947). A11 fractions were complex in structure, chiefly composed of units of D-arabin- ose, Demannose, Dugalactose, and L-rhamnose, and inositol in the lipid derivatives (Stacey and Kent, 1948). Isolations of components_from the organisms have been based generally on dilute acid and alkali extraction after the organisms were grown on synthetic media to exclude extraneous carbohydrate material. Polysaccharides from cells or filtrates were of similar nature (Stacey and Kent, 1948). Siebert (1950) reported that polysaccharides from unheated cells differed from polysaccharides of autoclaved cells, and that fractions from autoclaved cells of the same species varied from lot to lot. Free inosite instead of manninositose was found. Siebert isolated two different serologically specific polysaccharides from a filtrate. Polysaccharide I had a low molecular weight and was composed mainly of mannose and arabinose. Polysaccharide II was electrophoretically homogeneous, contained a small amount of lipid which may have been impurity, and a small amount of amino sugar. It was composed of a minimum of fifteen hexose units and contained no dextran, starch, or glycogen. Schaefer (1938, 1940) applied Landsteiner°s method (1919, 1920) of specific inhibition to study the polysaccharide 18 specific antibody with an active fraction from mycobacterial cells which was soluble in 50 to 85% methyl alcohol and an inactive fraction which was soluble in 50% methyl alcohol. The polysaccharide specific antibody was removed by excess antigen when the supernatant was measured by the comple- ment fixation test. Antigen suspensions prepared by adding the antigen drop by drop to saline were better than antigen suspensions prepared by adding antigen rapidly to the sa- line. The polysaccharide antigen was treated in three ways: heated in acid solution, heated in alkaline solution, and unheated. The results of the tests were identical with the antigens which were unheated or heated in acid solution. There was no reaction or a prozone reaction using the anti- gen heated in alkaline solution. Meyer and Pic (1936) removed the polysaccharide specific antibodies by adsorption to kaolin particles. The optimal kaolinuantigen complex required to remove the antibodies was determined by mixing antigen with varying amounts of kaolin. There was no adsorption of antigen to the kaolin with less than 10 mg of kaolin/m1 of antigen suspension. There was 80% adsorption with 10 mg of kaolin/ml of antigen suspension, 100% adsorption with 50 mg of kao- lin/ml of antigen suspension, and nonspecific adsorption with more than 50 mg of kaolin/ml of antigen suspension. With 50 mg of kaolin and 1.0 ml of antigen suspension, 98% of the specific antibodies was removed from the serum by 19 two adsorptions. To obtain the same results with one adsorp- tion, 8.0 m1 of antigen was necessary. Cross reactions were observed with antigen fractions prepared from M. tuberculosis, M. 232.239 11. £912; (Bacillus M), _!‘_l_. M and fl. leprae (Stacey and Kent, 1948; Iland, 1951). Tennent and Watson (1942) suggested that the poly- saccharides are genus specific rather than species specific. Some polysaccharides are haptenic (Stacey and Kent, 1948). They react with specific antibodies in dilutions of serum as high as 1:1,000,000 but do not elicit antibody formation. Polysaccharide II, a high molecular weight sub— stance, is reportedly a true antigen (Siebert, 1950). How- ever, Crowle (1958) suggested its antigenicity could be due to being complexed with a lipid. No tuberculoimmunity or- delayed hypersensitivity was induced in rabbits inoculated with either polysaccharide I or II (Siebert, 1950). Raffel (1946) inoculated guinea pigs with polysaccharides extracted from culture filtrates and from defatted tubercle bacilli. No tuberculoimmunity or delayed sensitivity was induced. , Tuberculophosphatides. A phosphatide is a lipid, which, when hydrolysed, yields fatty acids, phosphoric acid, an alcohol, which is generally glycerol, and a nitrogenous base such as choline or ethanolamine (Hawk, Osser, and Summerson, 1947). The extensive studies of tuberculophosphatides by Anderson and coworkers in the 1930's and l940°s were reviewed 20 by Crowle (1958). Anderson (1941) described the tuberculo- phosphatides as substances which were easily soluble in ether, precipitated from ether solution with acetone, and formed a colloidal solution in water. They contained 2.6 to 3.5% phosphorus in organic combination as glycerophos- phoric acid and as a phosphorylated polysaccharide or gly- ceride, and a small amount of nitrogen. When hydrolyzed, they yielded 33 to 40% water soluble compounds. These com- pounds yielded a small amount of inorganic phosphoric acid and a larger quantity of organic phosphoric acids similar in composition to glycerophosphoric acid when completely hydrolyzed with dilute sulfuric acid. The hydrolysed solu- tions also contained inositol, mannose, and a hexose which formed a typical glucosazone on treatment with phenylhydra- zine. Fatty acid components varied with each phosphatide. The saturated acids were chiefly palmitic acid. The un- saturated acids were chiefly oleic acid. In addition, every phosphatide contained saturated branched chain acids of higher molecular weight, which were oils at room temperature. Phosphatide fraction from M. phlgi (Chargaff, Pang- born, and Anderson, 1931),.M. tuberculosis (strain H37Rv) (Anderson, Lothrop, and Creighton, 1930), and Ef.222$§ (Anderson and Roberts, 1930) are very similar. As the phosphatide content of M? tuberculosis (strain H37Rv), 5. w, 31. 991.129 and M. M decreased respect- ively, the polysaccharide content increased. The following 21 results for each organism were reported: ‘M. tuberculosis 6.54% phosphatide, 0.97% polysaccharide; 33. M 2.26% phosphatide, 1.02% polysaccharide; E9.22!$§.1°53% phosphatide, 1.09% polysaccharide; and M. £h_l_e_i; 0.59% phosphatide and 3.90% polysaccharide (Chargaff, Pangborn, and Anderson, 1931). The composition of the phosphatides of heated bacilli differed from the composition of unheated bacilli (Siebert, 1950). The major difference was the absence of pure phthioic acid and the presence of a new substance, inositol glycerol diphosphoric acid, in the heated cells. It was not clear whether the differences were due to the use of different : strains or to changes caused by heating. No phosphatide or high temperature melting wax was found in human tubercle bacilli grown on Long's modified medium while other lipids seemed to be similar to those grown on standard medium. It has been the phosphatide from the classic frac- tionation of an ether-alcohol extract that has been studied chemically. Serologic and immunologic studies have been made of phOSphatides prepared from acetone-washed bacilli ex- ‘ tracted with methanol. Boquet and Negré (1923) prepared the original methanol extract known as antigene méthylique and used it as an antigen in serologic tests. The resistance inducing properties of antigéne methylique were reviewed by Crowle (1958). Boquet and Hegré (1923), Negré (1952), and other investigators have 22 proved that antigéne méthylique used prophylactically can increase the tuberculoimmunity of guinea pigs and rabbits but they indicate that such resistance is not necessarily equivalent to classic antitubercular immunity. Atypical mycobacteria. Atypical, anonymous or unclassi- fied mycobacteria exist other than those of the established species. They have been reported (Alvarez and Tavel, 1885; Griffith, 1916; Heaven and Bayne-Jones, 1931) since shortly after the discovery of the tubercle bacillus by Koch (1882). They have been isolated by many investigators. Until re- cently they have been largely disregarded as inconsequential saprophytes. Youmans (1963) cited several reasons for the tardy recognition of these organisms as causative agents of latent infection and pulmonary disease. First, after the isolation of M. tuberculosis by Koch (1882), the prev- alence of tuberculosis was so great that the relatively few cases which may have been caused by the atypical myco- bacteria were not apparent. Second, the failure of workers in diagnostic laboratories to cultivate and isolate rou- tinely the acid-fast bacilli present in the sputum or in- fected tissues of persons with tuberculosis. Third, the lack of pathogenicity for guinea pigs, and fourth, the presence of pigmented and non-pigmented saprophytes found in association with tubercle bacilli in sputum and gastric washings and in sputums of normal individuals. The atypical mycobacteria have been isolated repeatedly 23 and shown to be the cause of human disease (Tarshis and Frisch, l956a,b,c; Wood, et al., 1956; Runyon, 1959). Runyon (1959), jointly sponsored by the Veterans Administration and the National Tuberculosis Association, studied a large number of atypical mycobacteria and grouped them into four groups on the basis of pigment production and rate of growth. Group I, the photochromogens, produce no pigment unless exposed to light. Ten minutes' exposure during the period of active growth activates yellow pigment production during the sub; sequent incubation in the light or in the dark. Group II, the scotochromogens, produce yellow or orange pigment in : both light or dark and are more deeply pigmented if grown in continuous light. Group III, the non-photochromogens,' produce little or no pigment in the light or dark, and Group IV, the rapid growers, are generally non-pigmented. Group IV organisms require only three to four days for well iso- lated colonies to become visible on Lowenstein-Jensen medium at 37 C. The other three groups require ten to twelve days for visible colonies under the same conditions. Atypical mycobacteria similar to the Runyon groups of human disease have been isolated repeatedly from cattle and swine by members of the Michigan State University Tuber- culosis Research Project. The research project, sponsored jointly by the Animal Disease Eradication Division and the Animal Disease and Parasite Research Division of the United States Department of Agriculture, provides for investigating 24 the questions raised by the fact that many tuberculin posi- tive cattle when slaughtered show no gross lesions at ne- cropsy. Organisms isolated from such cattle have been stud- ied by means of various cytochemical and morphological char- acteristics, animal infectivity and allergenicity. Some are _M_. 2911-? Most are atypical mycobacteria. They were found to be heterogenous, highly variable and adaptable (Mallmann, Mallmann, and Robinson, 1964). Many of the iso- lants are classified as Group III organisms. These range. in virulence for calves, guinea pigs, chickens and rabbits from none to a virulence almost equal to that of g, bgzig. In addition, the Group III organisms isolated from swine tissues have little or no virulence for calves, but produce a disease in swine intermediate of that produced by M, bgyig and Er aging. Among the Group III organisms avirulent for calves, there are those which are believed to be identical to some of the Group III organisms isolated from human disease. These produce only intradermal lesions in calves. Others are undoubtedly non-pathogenic saprophytes. At present, there is no laboratory test which dif- ferentiates the virulent from avirulent Group III organisms. In the latter group, some strains produce some yellow color when the culture in a liquid medium is exposed unneces- sarily to light or upon prolonged incubation. These strains are called pseudochromes (Mallmann, Mallmann, and Robinson, 1964). (n h: m f c wt Ca Pe to Brc flat MATERIALS AND METHODS Cultures for inoculation. The cultures used to inoculate experimental animals are listed in Table 1. They were iso- lated from cattle, swine, soil and feed by members of the Bovine Tuberculosis Research Project, Michigan State Uni- versity, East Lansing, Michigan. Cultures are designated by the following coding system. Letters preceding the culture number refer to the method of isolation. “B“ refers to the pentane-enzyme method; ”M" refers to the pentane method followed by a disinfectant treatment; and no letter refers to the sodium hydroxide method. The letter "K" preceding the culture » number indicates the strain came from soil. The letters following the culture number refer to the tissues from which the strain was isolated; A = cervical lymph nodes, B = thoracic lymph nodes, C = mesenteric lymph nodes, D :1 carcass lymph nodes, E a lung, F a skin lesion, and H - Peyer's patches. The number following the letter refers to the year in which the culture was isolated; 0 - 1960, 1 a 1961, and 2 = 1962. Preparation of cultures for inoculation. Eight m1 Dubos Broth (Difco) with 0.5% dextrose were seeded with organisms and incubated at 35 C for approximately 14 days. The super- natant fluid was removed and the cells were resuspended in 25 26 TABLE 1. List of animals inoculated with mycobacteria Culture No. Source Mycobacterium Calf No. Swine No. 81-0 Swine Eb bovisl 20,81 2-1,2—2 2-4, 7-1 7-2,7-3 81-0 Swine M. bovis 26 The a't'Tk 1'1 led ) 130-0 Cattle M, bovis 6 Lab strain Chicken ‘M, avium 21 5-1,592 5-3 Lab strain Chicken M. avium 27 Theatekilled) 206-0 Swine ‘M, avium 10-5,lO-6 son-o Cattle III 1,66,67, 68,78,79, 80,87,88, 89 59,60,61, 62,74,75, 77,84,90, 91 51C-0 Cattle III (heat-killed) 29 620-0 Cattle III 3,44 sac-o Cattle III 4.45.63. 64,65,70, 71,73,82, 83,86 7lC-0 Cattle III 7,38,39 B783-0 Cattle III 10 I,' 1Where species is not indicated, organisms are atypical mycobacteria. Group. The Roman numeral indicates the Runyon 27 TABLE l.--Continued Culture No. Source Mycobacterium Calf No. Swine No. 93C-0 Swine III 16 lO7E-O Cattle III 13,40 152A1-l Swine III 19,41 167C-l Swine III 35,37 1-3,3-4 172C1-1 Swine III 23,30,31 1-1,164 6-1,6-2 6-3 173C-1 Swine III 24 186C-1 Swine III 34,36 3-1,3-2 193C2-1 Swine III 32,33 l-6,2-3 15D Swine pen III 161-4 192D Swine pen III 161-3 x37 Soil III 48 X41 Soil III 49,50 A Feed III 46,47 1128-0 Cattle Pseudochrome 17 128F-O Cattle Pseudochrome 8,56 52H-l Cattle Pseudochrome 11,57,58 613-1 Cattle Pseudochrome 14,55 Bll7B-O Cattle IV 15,52 Bll7B—O Cattle IV (heat-killed) 28 B124F-0 Cattle IV 5 4F-l Cattle IV 9 M7F—1 Cattle IV 18,51 BZS4F-l Cattle IV 25,53,54 28 Dubos medium so that the number of mg wet weight of cells injected in an animal was contained in 0.1 m1. Experimental animals. Experimental animals consisted of eight lots of animals as follows: Twenty-three calves (Lot I) were inoculated with cultures of M. 2931.5, M. M, Group III organisms (bovine origin), Group III organisms (porcine origin), pseudochrome organisms, and Group IV organisms. Four adult cows (Lot II), two from gross-lesion herds and two from no-gross-lesion herds, were not experi- mentally infected but were maintained under observation for approximately one year. Numerous tuberculin tests were administered. Of twenty-two swine (Lot III), three were inoculated with M. 2922-3: three were fed M. 2%, three were inocu- lated with M, gyggg, eight were inoculated with Group III organisms (porcine origin), and three were fed a Group III organism (porcine origin). Two swine were uninoculated. Two swine (Lot IV) were inoculated with Group III organisms (inanimate origin). Of thirty-three calves (Lot V), four were inoculated with heat-killed (1 hr, 100 C) cultures of £4;- 22322: 3. w, a Group III organism (bovine origin) and a Group IV organism. The remaining calves were inoculated with Group III organisms (bovine origin), Group III organisms (porcine origin), Group III organisms (inanimate origin), pseudochrome organisms 29 and Group IV organisms. Ten calves (Lot VI) were inoculated with Group III organisms (bovine origin). Nine calves (Lot VII) were inoculated with the same Group III organisms (bovine origin) as Lot VI. Nine calves (Lot VIII) were inoculated with the same Group III organisms (bovine origin) as Lots VI and VII. The experiments were performed over a period of time and were part of other experiments designed primarily to establish pathogenicity for swine (Ray, 1963), pathogen- icity for calves (McGavin, 1963), and route by which calves were susceptible to three Group III mycobacteria (McGavin and Goyings, 1963). Calves 1 through 6 from Lot I were injected intra- dermally at four sites, 0.1 or 1.0 mg per site, with a total of 2.2 mg wet weight of organisms. One mg was injected proximal to the carpus on the medial side of the left fore- leg and 1.0 mg was injected into the skin just distal to the hock on the medial side of the right hindleg. One tenth mg was injected distal to the carpus on the medial side — of the left foreleg and 0.1 mg was injected proximal to the hock on the medial side of the right hindleg. Calves: 7 through 25 from Lot I were injected intradermally with 1.0 mg wet weight of organisms into the lateral side of the left foreleg just proximal to the carpus. 30 The calves from Lot I died or were euthanized 37 to 86 days post inoculation. Calves 26 through 58 from Lot V, with the exception of 47 and 50, were injected intradermally with 1.0 mg wet weight of organisms into the lateral side of the left fore- leg just proximal to the carpus. Egl£_41 and £21: 52 were inoculated with a total of 10.0 mg wet weight of organisms by five routes. Two mg were given by each of the following routes: 'orally, intradermally at the same site as the other calves in Lot V, subcutaneously behind the right elbow, intramuscularly into the right gluteal mass and intraperi- toneally into the right paralumbar fossa. Calves 26 through 58 were euthanized or died 43 to 73 days post inoculation. Calves 59 through 69 were injected intradermally (ID) in the same manner as calves 26 through 58 with 1.0 mg wet weight of organisms. Calves 59 through 69 were divided into three groups of three animals. One calf of each group was euthanized two months after inoculation, two calves were euthanized four months after inoculation and one calf from each group was euthanized six months after inoculation. One calf died three months after inoculation. Nine heifers, 70, 71, 73 through 75, and 77 through 80, were inoculated with Group III organisms (bovine ori- gin). One mg weight of organisms was placed in a disposable 31 artificial insemination tube and a 10 m1 syringe containing 5.0 ml bovine semen was attached. The organisms and semen were introduced into the uterine cavity (IU) of heifers in induced estrus. The heifers were divided into three groups of three animals. Heifers 12; Zfl_and‘1§_were euthanized two months after inoculation, 12, 1g and 12 were euthanized four months after inoculation, and 1;, 11_and §Q_were euthanized six months after inoculation. Nine calves, 82 through 84 and 86 through 91, were exposed to an aerosol (AE) of Group III organisms (bovine origin). A calf was placed in an aerosol chamber and or- ganisms, suspended in 9.1% bovine albumin and 0.1% Tween 80 were sprayed into the chamber with an atomizer. Fifteen ml of culture medium containing approximately 1 x 108 or- ganisms/ml were sprayed into the chamber during a one hour exposure period for each calf. Three calves, one from each group, were to have been euthanized two months after inoculation, three at four months after inoculation and three at six months after in- oculation but, due to the severity of the disease, calves 83, _8__.:3_, §_6_, _9_9_, §_¢_1_, and 23; were euthanized prior to the scheduled time. £2l£_§2.was euthanized two months after inoculation, Eal£.§1_was euthanized four months after in- oculation and §§;£,§§_was euthanized six months after in- oculation. 32 Twenty-five swine were inoculated or fed 2.0 mg wet weight of organisms as follows: Type of Culture Swine Numbers Route M. 2225 2-1, 2-2, 2-4 ID 7-1, 7-2, 7-3 Oral M, gyggg 5-1, 5-2, 5-3 ID 6-1, 6-2, 6-3 Oral III 1-1, 1-3, 1-4 ID 3-4, 1-6, 2-3 ID 3-1, 3-2 ID 161-4, 161-3 ID 6-1, 6-2, 6-3 Oral They were injected intradermally on the lateral surface of the left anterior leg just distal to the carpus or were fed ration containing the organisms. Swine were killed 39 to 224 days post inoculation. At necropsy animal tissues were collected to be examined histologically. Results were recorded as follows: No Significant Lesions--no gross or microscopic lesions or only a granuloma at the site of inoculation; Primary Complex--1esion in the lymph node draining the inoculation site, may or may not have a skin lesion because skin lesion may have healed; Generalized-—lesions anywhere beyond the primary complex, i.e., disseminated in general circulation and generally in the lymph nodes draining the lungs. 33 Attempts were made at determining whether the les- ions were progressive or not on the basis of the criteria laid down by Feldman (1943) for guinea pigs. He stated that the signs of progressive lesions were: a) peripheral extension of the disease with daughter tubercles b) confluence of morbid tissue c) conglomerate tubercles d) slight to extensive necrosis Non-progressive lesions were characterized by: a) peripheral encapsulation without daughter tubercles in the peripheral zone of the capsule b) presence of noncaseating tubercles c) evidence of the transition of epithelioid cells to fibroblasts d) fibrosis e) calcification or bone where there are no signs of progressive tuberculosis Cattle and swine were tuberculin tested at inter- vals during the course of the experiments. The days post inoculation the cattle were tuberculin tested are recorded on Tables 5, 6, 7, and 8. Cattle were tested with 0.1 m1 mammalian tuberculin‘ injected into the caudal fold and 0.1 m1 mammalian tubercu- lin, 0.1 ml avian tuberculin,“ and 0.2 m1 Johnin"‘ in- jected into the cervical region. Results were read at 48' and 72 hr. 'Tuberculin, mammalian, intradermic produced for the Agri- cultural Research Service, U.S. Department of Agriculture. “Tuberculin, avian, intradermic, produced for the Agricul- tural Research Service, U.S. Department of Agriculture. “‘Johnin, intradermic, produced for the Agricultural Re- search Service, U.S. Department of Agriculture. 34 Swine which were inoculated with Group III organisms (porcine origin) and gs bgyig and uninoculated swine were tested with 0.1 ml avian tuberculin undiluted, 0.1 ml avian tuberculin diluted 1:5, 0.1 m1 avian tuberculin diluted 1:10, 0.1 m1 mammalian tuberculin undiluted, 0.1 ml mammal- ian tuberculin diluted 1:5, and 0.1 ml mammalian tuberculin diluted 1:10. Swine which were inoculated with'§,'gxigg, fed g, Eggig and a Group III organism (porcine origin) and inoculated with Group III organisms (inanimate origin) were tested with 0.1 ml avian tuberculin and 0.1 ml mammalian tuberculin. Avian tuberculin was injected into one ear, and mammalian tuberculin was injected into the opposite ear. Results were read at 48 and 72 hours. Collection of serum. Blood was collected prior to inocué lation and at different post inoculation times, as indicated in Tables 2, 5, 6, 7, and 8. After clotting, serum was removed and stored at ~20 C. Hemagglutination (HA) reagents and test. Saline: A stock solution of saline (final pH 7.2) contained NaCl 170.00 9 KHZPO 2.78 9 Na H964 11.30 g H2 qoSoado 2000000 ml For use the stock solution was diluted 1:10 with distilled water. Collection and sensitization of sheep erythrocytes: Sheep blood was obtained aseptically and put directly into 35 an equal volume of modified Alsever's solution. The mix- ture was stored at 4 C and used from one to ten weeks after collection. The erythrocyte suspension was removed from the flask, centrifuged, and was washed three times with buffered saline. Centrifugation was in an International Centrifuge Model UV at 550 X G for 15 min. .Modified Alsever's solution contained Dextrose 16.40 g Sodium citrate 6.40 9 NaCl 3.36 g Citric acid 0.44 9 H20 q.s.ad. 800.00 ml The solution had a pH of 6.1. It was dispensed in 200 ml quantities into 500 ml Erlenmeyer flasks and sterilized at 110 C for 10 min. Mammalian Tuberculin, License No. 107, Serial Num- bers 77 and 91, was supplied by the Animal Disease Eradica— tion Division of the United States Department of Agriculture. 01d Tuberculin was diluted 1:15 in buffered saline, and washed sheep erythrocytes were added, 0.1 ml per 6.0 ml of diluted OT. The suspension was incubated in a water bath at 37 C, 2 hr. After incubation, the suspension was: centrifuged at 550 X G for 4 min, and the supernatant fluid discarded. The cells were washed three times with buffered saline, and after the third washing, the cells were sus— pended in buffered saline at 0.5% concentration. The sen- sitized-erythrocyte suspension was not used beyond 24 hr. Inactivation and adsorption of serums: Serums were 36 inactivated by heating in a water bath, 56 C for 30 min. Washed sheep erythrocytes equal to 1/10 the volume of each serum was added and the mixture incubated in a water bath, 37 C for 30 min. After incubation the cells and serum were separated by centrifugation, the serum transferred to a clean tube, and the adsorption was repeated. The adsorbed serums were stored at -20 C. ;Serum dilutions: Beginning with dilutions of 1:2, 1:5, or 1:20, twofold serial dilutions of the adsorbed serum were made with buffered saline in 12 x 75 mm tubes. Serum and saline necessary to make the initial dilution in a total volume of 1.0 ml were placed in the first tube. After mixing the serum and saline no less than 10 times with a pipette, 0.5 ml was transferred to the second tube containing 0.5 ml buffered saline. This procedure was re- peated through the remaining tubes using a clean pipette after each transfer. The last dilution of 0.5 ml, from which further dilutions could be made if an end point were not reached, was stored in the refrigerator overnight. Controls of positive and negative serums and saline with senSitized and unsensitized cells were included. ‘Hemagglutination (HA) test: After three drops of 0.5% sensitized erythrocyte suspension were added to each of the serum dilutions, the tubes were shaken vigorously. Prelim- inary results indicated the most easily reproducible results were obtained by incubating in a water bath at 37 C 2 hr, 37 removing from the water bath and allowing to stand at room temperature for 2 hr and then at 4 C overnight. The tubes were removed from the refrigerator and allowed to warm to room temperature for 30 min. The tubes were swirled gently and observed under a 60 watt light bulb and against a white background. The reciprocal of the highest dilution of serum which caused agglutination was recorded as the HA titer. Kaolin-phosphatide (KP) reagents and test. . TME buffer solution: The TME buffer solution (Taka- hashi, 1962) was obtained from Takahashi for use in tests employing the Takahashi phosphatide-methanol solution. Sub- sequently, TMB buffer solution was made for other tests by the following formula: Tris (hydroxymethyl) amino methane 121.10 g Maleic acid (anhydrous) 9.80 9 EDTA (disodium ethylene diamino- tetracetate) 5.63 g Tween 80 0.05 g NaOH 3.75 9 H20 q.s.ad. 1000.00 ml The pH was adjusted to 6.6 by adding 1N NaOH. The buffer solution was dispensed to glass ampules. The ampules were fire-sealed and sterilized by heating at 100 C for 15 min. They were incubated overnight at 37 C, heated again in the same manner, and stored at room tempera— ture. The resulting suspension was usable for at least one year after preparation. Preparation of TME buffered saline: Buffered saline solution was prepared by adding one volume of TME buffer 38 to nine volumes physiological saline. The solution was good for one week. Preparation of phosphatide-methanol antiggg: Phospha- tide-methanol solution obtained from Takahashi (1962) had been prepared from g. bgyi§,(BCG) and fl, tuberculosis (strains H37Rv and Nakano). The organisms had been grown for eight weeks on Sauton medium and 60 g of bacilli was treated two times with 600 ml of acetone at 40 C, 3 hr each time. After drying the cellular residue was treated two times at the same temperature with 600 m1 methanol, 5 hr each time. The methanol extracts were combined, filtered in a Seitz filter, and evaporated in a nitrogen atmosphere to 200 m1. Two volumes of acetone were added, and the precipitate was col? lected by centrifugation, redissolved in warm methanol, and again centrifuged to eliminate impurities. Reprecipitation by addition of acetone was followed by drying in vacuo. Five-tenth m1 amounts of methanol solution containing 0.05 mg/ml of phosphatide from each strain of organism and 0.05- mg/ml of ovolecithin were distributed in brown ampules which were sealed and sterilized in the same manner as the THE buffer and kaolin suspension. A saline emulsion of phosphatide was used to treat the kaolin suspension. The saline suspension contained 7.5 gamma of phosphatide/m1. One part of phosphatide methanol solution was added dropwise by means of a 1.0 m1 pipette to 19 parts of buffered saline previously placed 39 in a flask and under constant agitation by means of a mag- netic stirrer. Preparation and sensitization of kaolin: Kaolin ob- tained from Takahashi (1962) had been treated with 5% HCl: and kept under constant agitation by means of a magnetic stirrer for 24 hr. The acid was removed and the kaolin washed repeatedly with deionized water. The kaolin-water suspension was filtered, and the kaolin was dried in a hot air oven at approximately 120 C and stored at room temper4 ature. Approximately 10.0 g of kaolin was heated in a cru- cible at 800110 C 2 hr. After cooling the kaolin was mechan- ically pulverized 2 hr in a porcelain mortar, 20 ml of H20 was added, and the suspension pulverized for 30 min. Suf- ficient water was added to yield a thick suspension which”. was then distributed in equal amounts into five 15 x 120 mm tubes. water was added to the tubes to a height of 10 cm, and the mixture allowed to stand 15 to 20 hr at room tem-' perature. The supernatant fluid was transferred to a cane trifuge tube and centrifuged for 15 min at 1240 x G. The supernatant fluid was decanted and discarded. The precipé itate was resuspended in water, 1.0 mg/ml. The kaolin sus- pension was dispensed into glass ampules, 6.0 ml/ampule, and sterilized by the same method as the TME buffer and phosphatide-methanol antigen. To sensitize the kaolin, one part of the standard kaolin suspension was added to two parts of the saline 40 suspension of antigen. The mixture was shaken thoroughly and incubated at 37 C for 30 min, during which time it was shaken every 15 min. The sensitized kaolin suspension was used only on the day of preparation. Inactivation and dilutions of serums: Serums were inactivated by chemical inactivation with EDTA (disodium ethylene diaminotetracetate) (Eastman Organic Chemicals). BDTAscontained in the TME buffered saline used for the di- lution of serums was sufficient to inactivate the complement. Beginning with initial dilutions of 1:10 and 1:20, twofold serial dilutions were made in THE buffered saline in the same manner as described for the hemagglutination test. Kaolin—phosphatide (KP) agglutination test: A modi- fication of the technique of Takahashi (1962) was followed. The sensitized kaolin suspension was added to each of the serum dilution and control.tubes, 0.1 ml per tube. The rack of tubes was thoroughly shaken and incubated 24 hr in a 37 C incubator in a plastic bag with a piece of damp- ened cheesecloth. Agglutination was observed with a con- cave magnifying mirror with the aid of a 60-watt bulb. The reciprocal of the highest dilution of serum which caused agglutination was recorded as the KP titer. Preparation of experimentalyphosphatide extracts: Phosphatide fractions were extracted from the following strains of organisms: g. bovis, fl. avium, and‘fl. phlei; 41 human origin P4 and P8 (Group I), P15 (Group II), P39 (Group III), and RIV (Group IV); Group III of animal origin, 186C-1 and 172C1-l (porcine), SlC-O (bovine), 266-1 (semen); and a Group III of soil origin, X9. With the exception of the g. 2.9.3.5. strain, grown in Dubos broth with 0.5% dextrose, all organisms were grown in Modified Sauton°s Medium. The Modified Sauton's Medium was prepared according to the fol- lowing formula and dispensed in 50.0 ml amounts in 250 ml Erlermeyer flasks. Asparagine 6.00 g Citric acid 2.00 g K2HPO4 1.50 g Ferric ammonium citrate 0.05 g MgSO4 0.25 g Glycerol 30.00 ml The ingredients were dissolved in distilled water, and the solution adjusted with concentrated NH OH to pH 6.2. Water 4 was added to a final volume of one liter. The solution was sterilized at 121 C, 15 min. An 80% glucose solution, slightly acidified with citric acid, was sterilized by fil- tration and 12.5 ml added aseptically to the first solution. The kaolin-phosphatide reagents and test were com; pleted as previously described with the following exceptions: The Seitz-filtered methanol solution was evaporated in a Buchler flashnevaporator under vacuum. Nitrogen was‘ introduced into the evaporator and flask containing the - methanol solution to remove the residual oxygen present. The final product was air dried and stored in the freezer at -20 C until used at which time it was dissolved in 42 methanol at a concentration of 0.15 mg/ml of methanol. Extracts prepared from M, gyigm, 186C-l, and 51C-0 evapor- ated to approximately 0.25 ml prior to sealing of ampules. The oven-dried kaolin was heated in a furnace at l 700 C 2 hr, and the heated kaolin was ground as finely as) possible using an agate mortar and pestle. It was then suspended in a 15 x 120 mm test tube, mixed and allowed to settle overnight (Takahashi and Ono, 1958). RESULTS Preliminary determinations of the HA test. Serum titers were two to fourfold higher when incubated for 2 hr at 37 C, room temperature for 2 hr and overnight at 4 C than when incubated for 2 hr at 37 C and overnight at 4 C; room tem- perature for 2 hr and overnight at 4 C; 1 hr at 37 C and overnight at 4 C; or overnight at 4 C. Readings were made at the end of each incubation period, i.e., 1 hr, 2 hr, 4 hr, and the following morning. When mammalian OT, avian OT, PPD-S, and Weybridge PPD (mammalian and avian) were used as antigens, mammalian OT yielded the highest titers with the positive control serum. 3 Sheep erythrocytes collected in Alsever's solution in our laboratory were less fragile than those commercially obtained. Serum HA and KP titers of uninoculated animals. Hemagglu- tination tests of serums collected from the calves and swine were not always zero. Calves 6, 1_3_, 16, 16, 11, 16, 66, _4_0_, 23’ 53., 2.5.9 5.8., 29.: 5.3.: 13.2: E: 15.: and Swine 3229 37—3., ELI» 6:1,‘6:6, 6:1, 6:6, 1:1, and 1:1_had serum HA titers rang- ing from less than 5 to 20. Titers of 20 or less were con- sidered negative. Four calves, 66, 61, 16, and 12, had pre-inoculation HA serum titers greater than 20. Calves 1 through 25 (Lot I). Serial HA serum titers of 43 44 23 calves inoculated with M. 2.91.1.5: M. m, Group III organisms (bovine origin), Group III organisms (porcine ori- gin), pseudochrome organisms, and Group IV organisms are presented in Table 2. Since the serums on which HA tests_ were performed were collected at different intervals from different calves after the calves were inoculated, serial titers are not always comparable. Initial or pre-inoculation and final HA serum titers, initial and final KP serum titers, and histopathologic find- ings are presented in Table 3. The following results are presented in groups inoc— ulated with a given type of organism. All calves had nega- tive HA serum titers prior to inoculation with organisms. All calves had negative KP serum titers except calves 16'and . .16. Calves 1g_and 16 had pre-inoculation.KP serum.titers of 40 and 80 respectively. Calves inoculated with M. bovis: 661£_6.(inoculated: with culture 130-0 which produced a local ulcer, disseminated lesions, progressive disease) had HA serum titers greater. than 20 after inoculation except the let day, at which time the titer was 20. The maximum HA serum titer was 80. The_ final KP serum titer was 80. 661£_16.(inoculated with culture 81-0 which produced a local ulcer, disseminated lesions, progressive disease) had HA serum titers of 32 or greater 37 days after inocu- lation with the culture. The maximum HA serum titer was 64. 45 TABLE 2. Serial tuberculopolysaccharide specific hema luti- nation (HA) serum titers of calves (1-25) inoculated with mycobacteria l l l 2 ggiggie sos-ol 51c-o 62D—0 68C-0 B124F-0 ‘§,,gg!1§ gaiier 1 2 3 4 5 6 3;?" _______. o - - 20 - 5 ' 7 64 80 4o - - so ‘14 ’ 128 so 40 5 - 80 21 32 4o 10 - 20 28 64 80 2o 10 - so - 35 32 4o 10 10 - 36 4°: 42 128 so 20 10 - 54 . 10 57 128 so 10 - 63 256 64 80 72 - 76 1° lGroup III--bovine origin. Group IV--bovine skin lesion origin. Days after inoculation of calves intradermally on the fore- legs, 1.0 mg and 0.1 mg wet weight at each of two sites per each amount. 46 TABLE 2.--Continued 333:? y, 20_Vi_s 6. £12.19. 71c-ol B7BB-01 10711-01 93c-05 152111-15. figifier 20 21 7 10 13 16 19 Days4 0 16V - - — 20 5 - - 30 16 so 10 20 so 5 37 64 20 20 so 10 so 10 45 64 20 so 10 4o - 52 64 so 20 so 10 20 1o 59 32 so 5 so - 10 ' “ 7o 64 so 10 10 10 1o 87 5 'ss 64 '.93 1o 95 so 99 100 101 10 107 so 10 4Days after inoculation of calves intradermally on the foreleg with 1.0 mg wet weight at one site. Group III--swine. 47 TABLE 2.--Continued figiggge 81178-02 4F-12 M7F-12 128F-06 52H-16 618-16 1128-06 fifiiier 19 9 l8 8 11 14 17 Days4 0 - - - - - 10 30 5 10 - 10 10 - 4o 37 1o 20 - 20 20 20 80 '45 - 20 - 1o 40 5 so 52 10 20 - 20 20 20 20 59 20 - - 4o 10 20 7o 10 1o - - 20 10 20 87 - 91 20 92 _ _ 94 - so 100 _ 101 40 107 10 108 - 6Pseudochrome mycobacteria. TABLE 2.--Continued 48 Culture 172C _15 number 1 173C-1 B254F-12 Calf number 23 24 25 Days4 0 _ - 5 _ - 24 - - ' 46 - — 55 - _ 62 - - 67 - - 69 - 10 20 49 .uconoud ma coanma mo scandasnamuuo n I won o>ansHuuoucH macaonummouuan u + .omdumao oouwamwucuu a + «doaunoumoun omummfiv nommwmdo o>wmmoumoum .haao mono: nmaha Udumucomoam odd one: he muoamoa a U a 4 «wade nooou naaha use: ca odd coauuamuoca mo open as muoamoa I 4 a m maoaumaauoaa mo open on» as quthdum e Mano no mdoamma UHQoumouUHa no mmowa on I Amz “dude nowadasuoufi on» unauHuuo moo: nuaha on» ma muoanua u um mqumEou hquHum on» ouchon muonshcu muOHmmH oouwdmumumm u now .uwomou hmoHonuom 3...? 32.3080 ngmhouam 03% mo room an anode: no: me a. 0 use. me o. a .mmoaouom on» no maameuoomwuca omumHsuouHH I Am: I ow ON ON ma quhoa I, Ana CA I cm I OH oImmhm I. - um cm I OH I h .oIUHb I - now I I 0v I v oIUmm I new ov I OH 0N m Olamm + now oma I om I N OIUHm . .. :Hmeo I now ov I mmm I H OImom QGH>OQIHHH I 4 a m oe om cm I am uflmuum .Il . n04 asw>m .z .. , coo own I so! we om .oIHm + coo oe I ,oe .. e enema meson mm ommmmdp uuonou Hmch ammuHGH Hmudm HMHuHuH Renaud H0385: mama .mwwemmoumoum «sooHonunm nous» me none» as memo..ouspeso massage nHHoHUdnouma sud: Hoouuaauocfl hmmlflp mmbamu Ho muuomuu. macaozuum one mwouwn Edwom Amxv mofiuenunonnnBHMDHRSphu.Admv coabmuwuuammofimm .m mqmH I qmz 0H 0H ow 0H ea oImNHH I Amz om ov OH I ea HImHm I Amz I I ON I AH HImNm I. 0.80ch + U a 4 OH I om I w clmmma lowdown I M Amz om OH I I em HIUth I . qmz ea on I I mm, HIHUNeH + on oH I on I ma HIHHmmowmowm hmoflosumm none» mx woven 4m Mano mumpasu ownuasu m N UOSGHHGOUIlom mdm<fi 51 The final KP serum titer was 160. Calf inoculated with M. avium: 6316,11 (inoculated with a laboratory strain of M, 31123 which produced a local ulcer, and lesions in the cervical lymph nodes, no progres- sive disease) had HA serum titers of 80 after inoculation except 37 days at which time the titer was 20. The final KP serum titer was 40. Calves inoculated with Group III (bovine origin) myco- bacteria: Calves _1_, 1, 6, g, 1, 1g, and 16 (inoculated with Group III organisms of bovine origin) varied widely in sero- logic and histopathologic responses. The results are pre— sented for each calf in numerical sequence. 6216.1 (inoculated with SOB—0 which produced a local ulcer, lesions in the pool of the anterior and posterior mediastinal and left and right bronchial lymph nodes, no progressive disease) had HA serum titers of 32 or greater after inoculation of the organism. The maximum HA serum titer was 40. The final KP serum titer was 40. 6616‘1_(inoculated with 516:0 which produced a local ulcer, disseminated lesions in many lymph nodes, progres- sive disease) had HA serum titers of 40 or greater after inoculation of the organism. The maximum titer was 80. The final KP serum titer was 1600 .Eél£.§ (inoculated with 62D-0 which produced a local ulcer, lesions in the right popliteal, right internal inguinal, and left prescapular lymph nodes, no progressive disease) 52 had maximum HA serum titers of 40 which occurred at 14 and 21 days after inoculation of the organism. Subsequent titers were negative. The final KP serum titer was 40. EEl£.$ (inoculated with 68Ce0 which produced a local ulcer, lesions in the pool of the anterior and posterior mediastinal and right and left bronchial lymph nodes, no progressive disease) had negative HA serum titers except the final serum. The final HA serum titer was 40. The final KP serum titer was negative. .EE££.Z (inoculated with 71Cm0 which produced a local ulcer, lesions in the left prescapular lymph node, no pro- gressive disease) had only negative HA serum titers. The final KP serum titer was 800 Calf 16_(inoculated with B78B-0 which did not pro- duce lesions or disease) had HA serum titers which were 80 at all times. The final KP serum titer was negative. 621£H11 (inoculated with 107Em0 which did not pro— duce lesions or disease) had HA serum titers which were negative at all times. Calves inoculated with Group III (porcine origin) myco~ bacteria: Serologic and histopathologic responses varied in calves 16, 12, 16, and 16 which were inoculated with Group III (porcine origin) organisms. 6616:16 (inoculated with 93C~0 which produced a local ulcer, lesions in the left prescapular lymph node, no progressive disease) had HA serum titers of 80 at 30 53 and 37 days and of 40 at 40 and 45 days after inoculation. Subsequent HA serum titers were negative. The final KP serum titer was negative. Calf 12_(inoculated with 152A ml which produced a 1 local ulcer, lesions in the left and right medial retropharyn- geal lymph nodes, progressive disease) had HA serum titers which were negative at all times after inoculation. The final KP serum titer was negative. l-l.and l73C—1 respectively which did not produce significant lesions or Calves 16 and 13 (inoculated with 172C disease) had HA and KP serum titers which were negative at all times. Calves inoculated with pseudochrome mycobacteria: In general, few or no lesions occurred in calves inoculated with the pseudochromes and serum titers were lower. 6616.6 (inoculated with 128F-0 which produced les- ions in the cervical lymph nodes and in the mesenteric lymph nodes, histopathologic evidence for disease progression inconclusive) had HA and KP serum titers which were nega- tive at all times. Calvesi11,‘1i, and 11 (inoculated with 52H71,i6lB-l and 1128-0 respectively which did not produce lesions or disease) had HA serum titers which varied. 6616.11_had HA serum titers of 40 at 45 and 59 days and of 80 at 94 days after inoculation. The remaining titers were negative. Calf 16 had only negative HA serum titers. Calf 11 had 54 HA serum titers of 40 at 30 and 101 days and of 80 at 37 and 45 days after inoculation. Other HA serum titers were negative. Final KP serum titers were all negative. Calves inoculated with Group IV mycobacteria: Calves 6, 2, 16, 16, and 16'(inocu1ated with B124P-0, 4F-l, Bll7B—0, M7P-l, and 254F-1 respectively which produced no lesions or disease) had HA serum titers which were negative at all times. Adult cows (Lot II). Serial HA titers of serums collected from cows 26, 21, 26, and 22,at one and two days prior and l, 2, 9, and 10 days after administration of tuberculin, pre and post tuberculin KP serum titers and histopathologic findings, are presented in Table 4. Cows 26.and 21, obtained from a no-gross-lesion herd, did not have fourfold increases in HA serum titers after tuberculin testing. Cows 26’and 22, obtained from a gross-lesion herd, had fourfold increases in HA serum titers after tuberculin testing. KP serum titers performed on one serum collected prior to injection of tuberculin and one serum collected after injection of tuberculin were similar to the HA serum: titers. Cows 26_and.21 did not have fourfold increases, cows 26_and.22 did have fourfold increases. At necropsy cows 26_and 21_did not have lesions or disease, and cows 22_and‘22 did have lesions and disease. Swine (Lots III and IV). Serial HA serum titers of 11 swine inoculated with g, bovis and Group III (porcine origin) 55 TABLE 4. Hemagglutination (HA) and kaolin-phosphatide (KP) serum titers and pathology reports of four cows before and after tuberculin testing Cow l l 2 2 number 96 97 98 99 Daysi HA titer -2 20 10 10 40 -l 20 5 20 80 20 20 20 40 3 20 20 160 20 10 2O 5 10 10 80 160 12 20 80 160 13 10 20 160 80 KP titer —2 20 20 10 2O 5 20 20 40 80 4 Pathology report NSL NSL Gen Gen Progressives disease — - + + éTuberculin positive cow obtained from no—gross—lesion herd. 3Tuberculin positive cow obtained from gross-lesion herd. 0 = day of caudal fold tuberculin test, 0.1 ml mammalian 4tuberculin. Pathology report: Gen = generalized lesions anywhere beyond the regional lymph nodes draining the inoculation site; NSL = no gross or microscopic lesions or only a 5granuloma at the site of inoculation. Progressive disease: + a generalized disease; - = encap- sulation of lesion if present. 56 organisms, three swine inoculated with g, EléEEJ three swine fed a Group III (porcine origin) organism, three swine fed 2, 26216 and two uninoculated swine are presented in Table 5. The HA and KP serum titers were negative in all animals prior to the injection of organisms. After the injection of organisms, the HA serum titers of the swine were higher than the HA serum titers found in calves.? Calves had relatively low HA serum titers and had little disease after inoculation with Group III (porcine origin) organisms. All of the swine inoculated with these organisms except 2:2 had lesions and HA serum titers which were at least 640 at some time during the testing period. Since serial HA serum tests were performed at vary- ing intervals of time, correlation between histopathology' and HA and KP¥serum titers are presented on the basis of a fourfold increase in HA and KP serum titers after tuber- culin testing. In general there was an increase in HA serum titers after tuberculin testing with decreases in titers during the intervals between tuberculin tests. Pre and post tuberculin HA serum titers, pre and post tuberculin KP serum titers, and histopathologic find- ings of 14 swine from Lot III, pre and post tuberculin HA 5 serum titers, histopathologic findings of eight swine from Lot III and two swine, which did not have serial HA serum titers, inoculated with Group III (inanimate origin) organ- isms from Lot IV, are presented in Table 6. S7 TABLE 5. Tuberculopolysaccharide specific hemagglutination (HA) serum titers of swine inoculated with mycobacteria Culture No. lg. bovis 172C -11 l67C-l Swine No. 2:1 2-2 2-4 1-1 1:4 1-3 3-4 Days2 _ l 20 - - - _ 8 40 20 20 20 40 20 4O 15 - 10 10 - 20 20 20 22: m 10 5 20 20 20 20 353 4o 20 10 20 20 160 38 80 320 20 80 80 44 160 20 320 320 640 640 640 52 160 160 160 640 160 320 1280 57 320 640 65 160 320 78 80 160 80 92 40 80 320 993 so 160 160 100 20 80 160 101 80 40 80 102 20 20 40 108 320 112 320 640 lWhere species is not indicated, organisms are atypical mycobacteria, Group III organisms (swine). Days after inoculation of swine intradermally on the fore- leg with 2.0 mg wet weight of M, bovis and Group III or- ganisms (swine). Days after inoculation swine were tuberculin tested. 58 TABLE 5.--Continued its? ”masts“ ixigzr 1-6 2-3 3-2 3-2 411 .4-2 Days: Days 0 - — 10 - — o 10 10 8 10 20 so 20 8 1o 20 15 . - 10 so 10 19 - - 22 10 10 5 10 32 40 _2o 353 1o 10 10 47 - - 38 ? 4o 20 1o 20 613 4o 5 44 t 640 320 640 640 70 4o 20 52 ' 1280 320 640 105 .80 4o 65 ' 160 117 40 lo 66 so 1313 so so 78 ' 160 so 140 so so 92 ‘ 160 40 161 so so 993 160 so 181 so so 100 160 40 196 40 40 101 so 40 2083 40 2o 102 so so 216 so so 108 160 113 1280 TABLE 5.--Continued gig? LII . avium igigzr 5-1 5-2 5-3, Days4 0 10 5 20 s é 40 640 10 19 : 10 80 2o 32 a 20 160 20' 47 10 so 10. 613? 20 so 20 7o 4 1280 1280 640 77 ? 160 105 _ 160 160 117 - so so 1313 160 so 138 5120 140 1280 161 . 320 181 160 196 40 2083 so 216 640 4 Days after inoculation of swine intradermally on the fore- leg with 2.0 mg wet weight of M, avium at one site. TABLE 5.--Continued fir €352? 1.2.1-. s 221—5 £3422: 6-1 6-2 6-3 7-1 7-2 7-3 Days5 0 5 - 10 1o 5 - 22 40 2o 20 so so so 36 10 5 20 4o 40 160 59 : ~ 20 20 4o 10 20 40 723' 40 so 1280 320 so 160 78 7 1280 640 1280 640 1280 320 92 i ' 640 2560 320 320 108 160 320 160 320 1365' 40 160 143 ‘ 640 1280 168 ‘ 640 320 177' 320 160 1963 160 160 204 320 1280 5Days after swine were fed Group III organism (porcine origin) and M, bovis. Swine were tuberculin tested 3 days previously. 61 .uuommwm ma coemoa mo coauoHSmamuuo u I muowmmmumowm ommmmfio wow 0>Hm3HucouuH mmoaonumaouman n “.monmomao omnwamwocom n + “mmmomao m>ammmwoowm .coaumasuoca mo open one um msoasumwm m made no mGOHmmH UHQOUmOHUHE no mmowm on n qmz mmuam GOHuMHSUOGH may ocfiuamwo moon edema or“ CH macemoa n um Axoadeou humaflua one odomon oumnzmcm muoamma nonaamwmcom u :00 nuHOQmu mmoaorumm .muwumou daasuwmndu umom when dais omuuoHHou Edwmm n umomm “ceasuwondu mo Goawumnufi on Howum mufiummu ceasuwmndu mo hop do omuumaaou Edwmm n mum w .omummn uflasuwmns» one: odw3m cowumHSUOCH umOQ when u w + :00 oma om ovm omH mm film omm 00H mm + now own oe oem om mm mIH HIUsmH + new omm 04 oem om mm wIH AhHHMEumU ovm oma mm Imwvuav + on oee oe omm om mm HIH HIHUNsH muesmIHHH omm om mm + :00 oma om omm 0H mm vIN + :00 om I owe om mm NIN Assameuou Imupufik + new cm I oma 0v mm HIN clam mfibon .2 omommwo phonon umom mum wumom Noun Hm Hogans Hones: ugh» vo>ammoumowm mhmoaocumm Honey mm woufiu «a Human odfl3m muspasu wusuasu maumpumnoome spa: nonnasuoufl mCflSm M0 muuommu NmoHonwwm one mumuflu Esumm Amxv moflnmmmmochCHHomx one Am .2 + um oem om ovm 0H mm NIm 00H om mm + um ovm om ovm m mm aIm HImeH + um 0mm om omm OH mm mam omma 00H mm . on see om oem owe mm was HINUmmH ommmmeo phonon #mom mum Nvmom Noun Hm Hogans umnfidc _udmu vo>Hmmmwooum mmmoaonumm woven mm usuau 4m when oufl3m-.wusuasu wubuasu UOSGHUGOUIIpm m4mon .2 omm oma mma oem omm mma ommmmao uwoamw umom mum Numom Noun Hm Hones: wonemc , waxy 0>Hnmmwmowm hmoaocuom wand» m2 woven <2 when OGHSm musuasu musuasu m H OUSGHHGOUllom mdmdfi 64 Swine inoculated ID with M. bovis: 621gg”2:1,'2:2, and.2:g,(inoculated with 81-0 which produced disseminated lesions, progressive disease) had fourfold increases in HA serum titers after a single tuberculin test. §E$22H3217 tuberculin tested two times, had fourfold increases in HA serum titers after both tests. All three swine had four— fold increases in KP serum titers after tuberculin testing. Swine fed M. bovis: 62166_1:2,and‘1:2 (fed 81-0 which produced disseminated lesions, progressive disease) had fourfold increases in HA serum titers after a single tuber- culin test. §E$22.Z:§J tuberculin tested three times, had fourfold increases in HA serum titers after all three tests. 4621gg,1:1’(fed 81-0 which produced disseminated lesions, progressive disease) did not have a fourfold in- crease in HA serum titer after a single tuberculin test. Swine inoculated ID with M. avium: 6g1gg_6:1,and.2:2 (inoculated with a laboratory strain of‘fl,‘gy1gg,which pro; duced lesions in the prescapular lymph node, no progressive disease) had fourfold increases in HA serum titers after both of two tuberculin tests. 62126_6:1.(inoculated with a laboratory strain of g,‘6!1gg which produced lesions in pre- scapular lymph node, no progressive disease) had a fourfold in- crease in HA serum titer after.one tuberculin teSt.. All three swine had fourfold increases in KP serum titers after tu— berculin testing. 65 Swine inoculated ID with Group III (porcine origin) mycobacteria: Swine 1-1 and 1-4 (inoculated with 172C -1 1 which produced lesions in prescapular lymph node, progres- sive disease in §El2£.l:l1 and disseminated lesions, progres- sive disease in 1:3) had fourfold increases in HA serum titers after tuberculin tests. §E£EEW£22 had one tubercu- lin test, 62123.1:1 had two tuberculin tests. ' 23122.1:2 and 6:2.(inoculated with l67C-1 which produced disseminated lesions, progressive disease) had fourfold increases in HA serum titers after one tuberculin test. .§!$22.$:23 which had two tuberculin tests, did not have a fourfold increase after the second test. .221ggn1:6 and.2:1 (inoculated with 193C2-l which produced lesions in the prescapular lymph node, progressive disease) had fourfold increases in HA serum titers after tuberculin tests. §E£22.l:§,had two tuberculin tests, and {62132H2:2 had one tuberculin test. §Ei22.§:l and 2:2 (inoculated with 186C-l which produced lesions in the prescapular lymph node, progressive disease) had fourfold increases in HA serum titers after one tuberculin test. .§E$22.§:ln which had two tuberculin tests, did not have a fourfold HA serum titer increase after the second tuberculin test. lSwine fed Group III (porcine origin) mycobacteria: Swine 6-1, 6-2, and 6-3 (fed 172C -1 which produced 1 disseminated lesions, progressive disease in Swine 6-3, lesions in prescapular lymph node, and progressive 66 disease in 6216616:1 and‘6:2) had one, two, and three tuber- culin tests respectively. §3$22.§:ln tuberculin tested once, had a fourfold HA serum titer increase.? 62122.6:2, tuberculin tested twice, had a fourfold HA serum titer in- crease only after the first tuberculin test. é!$22.§:§: tuberculin tested three times, did not have fourfold HA serum titer increases after any tuberculin test. Kaolin- phosphatide serum titers were not determined on these ani- mals. Swine inoculated ID with Group III (inanimate origin) mycobacteria: Swine 161-3 (inoculated with 15W, which did not produce lesions or disease) and 161:: (inoculated with 192W, which did not produce lesions or disease) did not have fourfold increases in HA serum titers after one tuber- culin test. These animals did not have KP serum titers determined. Uninoculated control swine: 62126 6:1 and 6:2.(unin- oculated controls) had three tuberculin tests. 62126“6:1 did not have fourfold increases in HA serum titers after any of the three tests. 6g1ggfl1:2 did not have a fourfold increase in HA serum titer after the first tuberculin test. There were fourfold increases after the second and third tests. Calves 26 through 58 (Lot V). Pre and post tuberculin HA serum titers, pre and post tuberculin KP serum titers, and histopathologic findings of calves from Lot V are pre- sented in Table 7. 67 .H non CH MHMU eonm ooHMHomHon EwHumanom .usnnonm MH nOHnoH mo GOHHuHanuuuo a I Asonnonmonm mudduflo now 0>HmsHusousH muOHonnuaouan u + AmmoomHu pouHHunouom I + .umdmnnu amenonmonm .uoHansuouH mo oan on» no mHOHsuunm m hHuo no nuOHmmH UHQOOnonUHEm. no muonm o: a Hmz AmuHm COHusHsuocH on» mcHdeno moon nQBhH on» dH muonmH u um Aondeou mnMEHnd on» osoamn mnonzhud mGOHnOH ouuHHgnwuom a new Innonon hUOHonuam .mcHumou uHHsunonsv anon when Hm on m oouuoHHou annwn n whom Adesu Inonsu mo GOHnuonuH on nOHnd muHunou nHHsunonsu no hot so omuuoHHou Bsnom u and .oounon GHHsunonsu onus mm>Hmu GOHuuHsuonH umom ewwom .sms c. on uo>nouun cm unho 6cm ee memo .wconudeuwum .ucmHmz no: Ema o.H 29H: mmHmnom on» cH MHHmEnmomnuuH omumHaoonH mo>Hmu HH¢ G H I Hmz cm I owH I #4 mm I qmz cm I cmNH I ee mm HImommH I Hmz cm I omH ov we Hm I nmz cm I eve oe ee om HIHUNAH munsmIHHH AomHHHxv _ uHmHno I Hmz ON I om I we mm OIUHm mCH>omIHHH I _ Hmz om I om I we mm oImsHHm ApoHHan >H nooHH HAP I Hmz om I 00H 04 we em and ESH>0 . nusHHHAP I Hmz om I omH I we mm OIHm _ mnbon..2 mmmomHo unoaon umom one numom mend Hm nonasc nonaan . Hunk» m0>Hnmonmonm hvmmoHonumm nouHu m2 nouHu 42 «when MHmU onsuHsu “unnuHsu I"! : dHnouquOUHE :qu ooumHsuocH Am mI c mo>HoU.me munoaon NmmHonyna one mnouHu esnom Amxv ooHuannon IGHHomx one Admv GOHnmanmwwmmmmm Ab12umda 68 I Am: ow m me mm Hlmemmm I an: m ov me mm clmeHHm I Hmz 0H m me Hm Hlmbz >H I Hmz I om om om Ne om Hex I am: om cm cm cm me me Hex .4 I qmz cm I oe om, we we emx HHowIHHH I Hmz om ow owH omH me be 4 :2w: . I _dmz cm cm can con we we 4 someHHH + coo om I ovm om mm me mOIUmm + now. ow om omH ov Nm we melamm + cow om om omH omH mm mv mOIUHm I on em I omH om mm Nv OIUHm I Hmz om om ov 04 we ov moImHOH I Hmz I om om. I we mm nOIUHe sHmHno I Hmz ON I ON I we mm OIUHe ouH>omIHHH I qmz cm I cm I me He eHIH<~mH I Hmz om I omH om . we em I Hmz cm I own 04 we mm HIUsmH I Hmz cm I cm I we on I Hmz cm I I I en en HIUmmH omoomHo anomon umom one mumom monm Hm nonauu nonesu .duahu mo>Hunonmonm emmoHonuom nouHu mg nouHu <2 women WHoU onsuHsU . .onpuHsu coscnucoUII.e unmHunonmonm ehmoHosnom nouHu mm nonHu.4m «when MHoU onansu . ._mnnuH:U uonanuouII.s.mamco mGOHmoH oouHHonocom u cow .mnHumou GHHsunonsu umom wasp eHIs oouooHHou Esnom n umom .ucomond MH.mmeono GOHuoHsnaouso u I Aomoome ooNHHdnocom u + .tho oooa :QENH UHnoucomoB I opoz U Aano oooc SQENH nonmoumonQ now o>HmzHUaousH hmoHosuomouch + AGOHmmonmond omoomHo .«ommomHo o>Hmmonmonm e u oooz a ACOHuoHsuouH mo ouHm onu no osoHsnonm o mHso no mGOHmoH UHmoomonUHE no mmonm o: u Hmz AouHm.:0HuoHsuocH on» mchHono ouoc sasmH onu CH mGOHmoH u um “unomon hmoHonpom ACHHsum Inonsu mo COHuuoth on nOHnm muHumou CHHsunonsu mo moo co oonuoHHou esnom n onm .ooumon CHqunonsu ono3 mHoEHno GOHuoHsuosH noon madam H I um 00H Cd 0% v0 + 20$ 0v mm mm + 20$ omH 0v 0? mm OIUmm ONm 0v mmH omH 0v NOH I now om I we mm own 0% NOH H coo 03 cm we co H coo oee I we 8 + G00 020G mm OIUHm .Q.H omoomHo nnoaon Numom wonm Hm nomads nonesc. . WW>Hmmonmonm mamOHocuom nouHu 42 Human MHoU onauHsu oufiom _mmmHno ouH>on mo oHnouuonouxa.HHH msonu mo adv Hononnla so on vomoaxo nOthPocHnousIonuuwdnaHv HHoEnooonu:H couoHsvbflfl mo>Hou mo nanomon oHosqu,oco mnouHu Esnom Acme COHuosHusHmfimmmmfl mm Wanda 75 Q ommH owe mmH ovm omH mOH + now own 04 me He omH om mOH + coo cm 04 Ime on + now omH I me me Clumm omH omH mmH omH oe mOH + cow oe om me he owe omH mOH + sow omm ov me me + um om om me on QIUHm .D.H own omH mmH omH omH NOH I Hmz omH ov we we owe omH NOH I Hmz ow I we so I Hmz omH ov we we Olmom omH oe mmH omH omH NOH ouoouHo unoaon numom Nona Hm nonamc nonasu o>Hnmonmonm hmoHonudm nouHu <1 when MHmU onansu ousom m ooscnucooIIwe numHnmonmonm mhmoHonuom nouHu <2 Hmhoa MHMU onsuHsu oudom U09fifi¥d00llom mqmdfi 77 6616 66 had a fourfold HA serum titer increase only after the first of three tuberculin tests. 6616.61_had fourfold HA serum titer increases after two tuberculin tests. 6616.66_had a fourfold HA serum titer increase after one tuberculin test. Heifers inoculated intrauterine (IU) Lot VII). Pre and post tuberculin HA titers of serums and histopathologic findings of nine heifers, inoculated with Group III organ- isms (bovine origin) SlC-O, 68C-0, and SOB-0 are presented in Table 8. Heifers 74, 75, and 77 inoculated with SlC-O: Heifer 1:,(which produced lesions in the mesenteric nodes, uterus, and intestine, progressive disease) had a fourfold HA serum ?titer increase after one tuberculin test. Heifer 16.(which had disseminated lesions, progressive disease) had fourfold HA serum titer increases after two tuberculin tests. Heifer. 11_(which had disseminated lesions, progressive disease) had a fourfold HA serum titer increase only after the sec- ond of three tuberculin tests. Heifers 70, 71,L and 73 inoculated with 68C—0: Heifers 16, 11, and‘16 (which produced disseminated lesions, pro- gressive disease) had varying HA serum titer increases after tuberculin testing. Heifer 16'had less than fourfold HA serum titer increases after two tuberculin tests. Heifer 11.had four- fold HA serum titer increases only after the first and second 78 of three tuberculin tests. Heifer 16.had a fourfold HA serum titer increase after one tuberculin test. Heifers 78, 79, and 80 inoculated with SOB-0: Heifer 16 (which had a microscopic lesion in the body lymph node, no progressive disease) had a fourfold serum HA titer in- crease after one tuberculin test. Heifer 12,(which had a microscopic lesion in the mesenteric lymph nodes, no pro- gressive disease) did not have fourfold HA serum titer in— creases after two tuberculin tests. Heifer 66.(which did not have significant lesions or disease) had fourfold HA Serum titer increases after the second and third of three tuberculin tests. Calves of aerosol exposure (AE) (Lot VIII). Pre and post tuberculin HA titers of serums and histopathologic findings from nine calves, inoculated with Group III organisms (bovine origin) SlC-O, 68C-0, and SOB-0, are presented in Table 8. Calvgsg64, 90, and 91 inoculated with SlC-O: [6616H26 died prior to collection of the post tuberculin serum. A £616,6g,(which had disseminated lesions, progressive dis-' ease) and'6616.21 (which had lesions in the right and left bronchial lymph nodes, and anterior mediastinal lymph nodes; progressive disease) did not have fourfold HA serum titer? increases after one tuberculin test. 6Calves 81, 83, and 86 inoculated with 68C:0: All three calves had disseminated lesions with progressive disease.? Calves 61_and 66 had fourfold HA serum titer increases after 79 one tuberculin test. £2$£.§§.did not have a fourfold HA serum titer increase after one tuberculin test. Calves 81, 88, and 89 inoculated with SOB-0: Signifi- cant lesions or disease was not produced in any of these calves. 6616 62 had a fourfold HA serum titer increase after one tuberculin test. 6616 61 did not have fourfold HA serum titer increases after two tuberculin tests. .221: 66 had a fourfold HA serum titer increase only after the third of three tuberculin tests. Kaolin-phosphatide serum titers of calves and swine inoc- ulated with mycobacteria usipgphosphatides extracted from various mycobacteria. Preliminary titrations were performed with reagents prepared in this laboratory and with Takahashi's reagents using serums from a cow inoculated withl§,'ggy1§ and a pig inoculated with 6, 62166, Comparable results were obtained with all combinations of reagents. Sample results are shown in Table 9. Kaolin-phosphatide titers of serums from four swine and five calves which were tested with phosphatide extracts of the following strains of organisms, 5. m, 6. 66216, g. hlei, l86C-l, 172C -1, 266-1, p39, 94, 98, p15, x9, 1 and RIV, are presented in Table 10. Serums from 62126_16:6_and 16:6, which had been inoculated with _Ig. 311% (206-2) had the following results: KP serum titers of éfliflé 16:6 were equal to or greater than 80 with six of the 13 extracts. Titers of 40 were obtained with two of the extracts. Titers with the remaining five 80 'mssns 9. Comparison of kaolin:phosphatide (KP) serum titers using Takahashi's reagents and_prepared reagents Reagents Bovine serum Swine serum T Kaolin‘ T Saline T Antigen 160 160 R Kaolin“ T Saline T Antigen 160 160 R Kaolin tR Saline T Antigen 160 160 r T Kaolin R Saline T-Antigen 160 160 R Kaolin R Saline R M.avium Antigen '40 -T Kaolin R Saline R H o aVium Antigen 4O .T a TakahBShi o "R a Robinson. .Aoconoz out 1. >mhmm mcHonumv anoHsunonsu Am can Acumv.nH>on.ufl.Bonm oonodond somano HammnmxoaH 8 . . , _ om oe oe om om oe ov ee om HHH HHomImx on on ov om om oe cm as om HHH dossmImmd I I on I oe on em I I -HHH cosomIHIeoN I cm I I cm I om cm I Hnn ocn>omIquHm on ‘oH I I I I on on I HHH ocnzmInIHu~eH oe .oe oe I om om cm I I HHH ocnzmIoIUemH om cm I cm on on cm I I ssn>m_um. oe cm on om cm I cm I cm wn>on mm cm own as con con anchorages noun» an moonnordhose . I I I I A nonasu we - mm H m m 0H m on Hm Hm om e m Hounc< . I IH I I nonfisn oImom o uHm H omen m mom nos o no ousunoo HHH Ends!“ om MHKVOQ om. .mQNU. .onansU oHnoqunouma nsOHnw> Bonn vouuonvxo nomduonmnonnmch: oHnoHUmnbUNE nuHa OouoHsoocH oqum Odo nobHoU mo mnouHu Eanom «may oUHuochonthHeoux .OH uqmH cossmI>ne on cm as oe om cm em on em .ousceoueouIHoncm am on on on on oe om om cm I HH cosomImHe om cm om om om om om om cm H coesmImd on on oe om cm as om oe cm H cossmIed noun» as “oonuocehocd I I I I nomads: we mm H e e on m on Hm Hm cm 4 N Hosnu< I IH I I none—Sc oImom 0 one H omen N mom nos 0 Hm ousonso .I. omen”. HHH ssn>o z wH>on «unease UTSGHHGOUII. OH Amanda. 83 extracts were negative. A titer of 160 was obtained with Takahashi's antigen. The KP serum titer with the homologous 11. 23.3.9.2 extract was only 10. 611.116 19:6 had KP serum titers equal to or greater than 80 with five of the extracts and Takahashi's antigen. Titers with the seven remaining ex- tracts were negative. The KP serum titer with the homolo- gous 11'. m extract was 20. ' Serum from 6613.: 6:1, which had been fed 172c1-1,‘ J? had a KP titer of 80 with the P8 extract, titers of 40 with six extracts, and negative titers with six extracts. The KP serum titer with the homologous extract was negative. Serum from 6:76.66 3:1, inoculated with 5. 2.9132 (81-0), had titers equal to or greater than 80 with the homologous 11. 221E extract and six other extracts. Titers with the six remaining extracts were negative. With Taka- hashi's antigen the KP serum titer was 160. 66:16 11, inoculated with 6. 91165,. had KP serum titers of 40 with three of the extracts and with Takahashi's antigen. The KP serum titer with the homologous g. M extract was 20, with the other nine extracts negative. _C_:_a_2_|._f_ 16, inoculated with _11. 29.3.12 (81-0), and gag 61, inoculated subcutaneously with‘g,‘6611§ (81-0), had KP serum titers of 80 with three and four extracts respec- tively. 6616_16_had a KP serum titer of 20 with the homol- ogous extract. 6616 61 had a KP serum titer of 80 with the homologous extract. Serum from Calf 16_had KP serum 84 titers of 40 with three extracts and negative titers with seven extracts. Serum from Eéi£.§l had a KP serum titer of 40 with one extract and negative titers with eight ex- >tracts. A titer of 160 was obtained for serum from 6616 16'with the Takahashi antigen. The test with serum from ‘6616561Dwas not performed with the Takahashi antigen. ? 6616:62, inoculated with SlC-O, had KP serum titers of 80 with the homologous extract and three other extracts, 40 with two extracts and negative titers with seven extracts. 6616 66, inoculated with 508-0, had a KP serum titer of 80 with one extract, 40 with two extracts, and negative titers with 10 extracts. The KP serum titer with the homologous extract was negative. Kaolin-phosphatide serum titers of calves 30 throug6_62 inoculated with mycobacteria. Kaolin-phosphatide titers of serums from calves 30 through 50 with phosphatides ex- ? tracts prepared from cultures of _M_. 1292.6, 1!, 63162, 186C-0, 152ci-1, 51c-o, 266-1, 939, x9, and Takahashi's antigen are presented in Table 11. ? Maximum KP serum titers equal to or greater than 160 were obtained for serum from‘6616D66,with extract pre- pared from 266-1 (semen origin) and for serum from‘6616'63; with extract prepared from X9 (soil origin). These organ- isms were identified as avirulent Group III organisms. .221: 66 had been inoculated with a Group III (porcine origin) organism (186C-l), which did not produce significant lesions or disease. Calf gg_had been inoculated with a Group III 85 .H you s« MHIU Ionm poudHowHon adeduno .sHmHno HHomIIHHH anonmm .chHno coasnllHHH asonwAV .chHno coaomIIHHH anonwm .AocoxozN one >m>mm mchnumv mHmoHsunonnu Am one mH>on.gfl.aonm oonoaona comHuco HnmocoxosH 1 II on as es on I on I oe on He mnIH<~mH I oe on on on I I cm on em HIHUAen oe on con I on I I on on em nIUmmH es on I I on on on on on mm HIHUAeH on as oe . ce, oe co oe on on em HIUemH oe oe oe . I on on I cm on mm on on, oe .- I on on on on on mm HINUmmH oe cm ,on . I , on on I . on on Hm . on on on on on on I . on on HIHUNAH ocnsmIHHH emx one nIeom oIUHm HIHUNAH Huoomn ssn>o am.nn>ob um.He nonssc umnsoc mesh IIIIhIIIImr nHoo ,ousunso ousuHou QOHuonoQonQopHuonmmonm Ir? IIIroHnouuuQOUNE_mSOHno> Eonm oopuonuxomooHuonmnonmmCHm5.oHnoPDwn Iouha nqu oouoHsuocH mo>HoU mo mnonHu Esnom Amxv ooHuonmmonuinfiabox .HH mqmomIHHH «an meme mnIoom quHm HIHUNAH HIUmmH son>o.nm nHwob in He noses: . nodes: was» cOHuonoQona ooHnonmmonm. mHoO onspHsu onansu Dr .. ILItIr oossnucoUII.HH mam