Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
INFORMATION TO USERS This reproduction was made from a copy of a document sent to us for microfilming. While the most advanced technology has been used to photograph and reproduce this document, the quality of the reproduction is heavily dependent upon the quality of the material submitted. The following explanation of techniques is provided to help clarify markings or notations which may appear on this reproduction. 1. The sign or "target" for pages apparently lacking from the document photographed is "Missing Page(s)". If it was possible to obtain the missing page(s) or section, they are spliced into the film along with adjacent pages. This may have necessitated cutting through an image and duplicating adjacent pages to assure complete continuity. 2. When an image on the film is obliterated with a round black mark, it is an indication of either blurred copy because of movement during exposure, duplicate copy, or copyrighted materials that should not have been filmed. For blurred pages, a good image of the page can be found in the adjacent frame. If copyrighted materials were deleted, a target note will appear listing the pages in the adjacent frame. 3. When a map, drawing or chart, etc., is part of the material being photographed, a definite method of "sectioning" the material has been followed. It is customary to begin filming at the upper left hand corner of a large sheet and to continue from left to right in equal sections with small overlaps. If necessary, sectioning is continued again—beginning below the first row and continuing on until complete. 4. For illustrations that cannot be satisfactorily reproduced by xerographic means, photographic prints can be purchased at additional cost and inserted into your xerographic copy. These prints are available upon request from the Dissertations Customer Services Department. 5. Some pages in any document may have indistinct print. In all cases the best available copy has been filmed. University Microfilms International 300 N. Zeeb Road Ann Arbor, Ml 48106 N 1320110 MAPOTHER, MARY ELIZABETH IN VITRO INTERACTION OF MYCOBACTERIUM AVIUM WITH INTESTINAL EPITHELIAL CELLS THE UNIVERSITY OF ARIZONA University Microfilms International 300 N. Zeeb Road, Ann Arbor. MI 48106 M.S. 1982 IN VITRO INTERACTION OF MYCOBACTERIUM AVIUM WITH INTESTINAL EPITHELIAL CELLS by Mary Elizabeth Mapother A Thesis Submitted to the Faculty of the DEPARTMENT OF ANIMAL PHYSIOLOGY In Partial Fulfillment of the Requirements For the Degree of MASTER OF SCIENCE In the Graduate College THE UNIVERSITY OF ARIZONA 1 9 8 2 STATEMENT BY AUTHOR This thesis has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under the rules of the Library. Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. SIGNED APPROVAL BY THESIS DIRECTOR This thesis has been approved on the date shown below: / J. G(^So)iger rofessor of Veterinary Sciences ACKNOWLEDGMENTS I would like to extend my appreciation to Dr. J. G. Songer for his encouragement, guidance, and often necessary sense of humor. To the entire staff of the Veterinary Science Department, my sincere gratitude for their assistance and patience. For his valuable time and expertise concerning computer analysis, I wish to extend a very special thanks to Dr. L. M. Kelley of the Department of Microbiology. And, to my parents, Richard and Francis Mapother, my greatest appreciation for their continuous faith and love; without them, this thesis would not be possible. TABLE OF CONTENTS Page LIST OF ILLUSTRATIONS v LIST OF TABLES vii ABSTRACT viii 1. REVIEW OF LITERATURE 1 2. MATERIALS AND METHODS 25 Experimental Design Bacterial Cultures Cell Cultures Antiserum Preparation Method of Assay Staining Techniques Microscopy Statistical Analysis 3. 25 25 26 28 30 35 37 37 RESULTS AND DISCUSSION 40 Kinetics Studies Host Cell Effects on Uptake Bacterial Effects on Uptake 4. 40 48 54 CONCLUSIONS 67 LIST OF REFERENCES 68 iv LIST OF ILLUSTRATIONS Figure 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. Page Kinetics of the Interaction of M. avium Serotype 2 with HIE Cells 41 Percent of M. avium Serotype 2 Shown to be Intracellular when Associated with HIE Cells 42 Percent of M. avium Serotype 8 Shown to be Intracellular when Associated with HIE Cells 44 Percent of M. avium Serotype 10 Shown to be Intracellular when Associated with HIE Cells 45 Kinetics of the Interaction of M. phlei with HIE Cells 46 Kinetics of the Interaction of M. smegmatis with HIE Cells 47 Difference in Uptake of Autoclaved and Viable M. avium Serotype 2 by HIE Cells 55 Staining Characteristics of Methanol-Fixed and Unfixed HIE Cells Infected with Autoclaved M. avium Serotype 2 56 Difference in Uptake of Formalin-Killed and Viable M. avium Serotype 2 by HIE Cells 57 Staining Characteristics of Methanol-Fixed and Unfixed HIE Cells Infected with Formalin-Kilied M. avium Serotype 2 59 Difference in Uptake of M. avium Serotype 2 Irradiated with Ultraviolet Light for 0 to 60 Seconds 60 Staining Characteristics of Methanol-Fixed and Unfixed HIE Cells Infected with M. avium Serotype 2 Irradiated with Ultraviolet Light for 0 to 60 Seconds 61 v Figure 13. 14. 15. Page Interaction of M. avium Serotype 2 Suspended in Various Concentrations of Homologous Antiserum with HIE Cells 63 Interaction of M. avium Serotype 2 Suspended in Bovine Milk or in MEM with HIE Cells 64 Interaction of M. avium Serotype 2 Suspended in Bovine Colostrum or in MEM with HIE Cells 65 vi LIST OF TABLES Table 1. 2. 3. 4. Page Effect of Cytochalasin B on Interaction of M. avium Serotype 2 with HIE Cells 49 Effect of Dibutyryl-cAMP on Interaction of M. avium Serotype 2 with HIE Cells 51 Effect of lodoacetate on Interaction of M. avium Serotype 2 with HIE Cells 52 Effect of 2,4-Dinitrophenol on Interaction of M. avium Serotype 2 with HIE Cells 53 vii ABSTRACT An in vitro model was developed to study the infection of cultured Henle intestinal epithelial (HIE) cells with pathogenic Mycobacterium avium and saprophytic mycobacteria. Results indicate that uptake of pathogenic M. avium occurred after 2 to 3 hours of incubation. Saprophytic mycobacteria did not attach to or penetrate the host cells of this model. Conditions necessary for uptake of M. avium serotype 2 by HIE cells include viability, expression of surface molecules, and normal metabolic activity of the tubercle bacilli. Furthermore, results indicate that the mechanism of uptake is an endocytic process induced by virulent mycobacteria. viii CHAPTER 1 REVIEW OF THE LITERATURE Swine tuberculosis is a disease problem that has affected the pork industry of the United States since the early 1900's. 1 OR The prevalence of tuberculosis in swine has been estimated at 0.85% of all swine slaughtered under federal inspection, 109 representing an almost 5 35 $6 million annual loss to the pork industry. ' The incidence of tuberculosis in swine is related to the opportunity for direct or indirect contact with tuberculous primates, cattle, avian species, and numerous environmental sources. Over the years most tuberculous lesions in U.S. swine have been found to be of avian origin.^' ^ U.S.D.A. statistics have shown that chickens in the north-central region of the United States have the highest per centage of condemnations due to lesions of tuberculosis.^' ^ This coincides with reports that states of the west north-central and east north-central accounted for more than 83% of swine condemnations and for more than 63% of carcasses retained for tuberculosis in 1971.^' ^ Nationally, the incidence of swine lymphadenitis has declined. 6 Most of this decline is largely attributable to an increase in the prac5 tice of maintaining all-pullet farm flocks among poultry producers. ' 20 Control of tuberculosis among cattle, through tuberculin testing and slaughter of reactors, is also thought to have decreased the loss among hogs. 5 Of the 31,843,375 cattle slaughtered under federal 1 2 inspection in 1972, only 246 carcasses (0.0008%) had tuberculous lesions. In contrast, of 83,126,396 swine slaughtered during the same year, 712,227 carcasses (0.85%) had lesions suggestive of tuberculo5 20 sis, nearly 1000 times more than were found in cattle. ' The above figures indicate that tuberculosis may no longer be considered such a serious problem in swine in the U.S., but the decrease of the disease has not been as pronounced as for bovine and avian tubercu losis. In 1972, the Food Safety and Inspection Service of the U.S.D.A. instituted new rules and regulations concerning tuberculous lesions in swine. 57 Before 1972 when these granulomatous lesions were found at slaughter, they were excised and discarded, and the remainder 5 20 of the carcass was processed as usual. ' Following new regulations, swine carcasses, and/or parts, are labeled as condemned, passed for cooking (PFC), or passed without restriction for human food. 57 The entire carcass is condemned when the character of the lesions is not indicative of a localized condition. Portions of carcasses and whole carcasses are labeled PFC if they reveal lesions indicative of a localized condition. Such carcasses or parts must be cooked at 76.7°C for 30 minutes after removal of the infected part to be considered fit for human consumption. Portions of carcasses and car casses of swine are passed without restriction provided the carcass is found free of lesions or when lesions are found confined to one primary site of infection. demned carcasses is obvious. The economic devastation due to con Cooked carcasses lose about 2/3 of 3 their original value. 5 ' 20' 106 This loss is felt by the packing industry and the consumer, but mostly by the pork producer, since he not only obtains a lower price for his hogs, but may lose his market altogether. 5 ' 2 ®' ^ Etiology Historically, the etiologic agents of tuberculosis in swine have been the same agents which have caused tuberculosis in the animals with which swine have had contact. Before the start of attempts to eradicate both human and bovine tuberculosis, isolations of tubercle bacilli from lesions in swine were often shown to be of either bovine (Mycobacterium bovis) or human (Mycobacterium tuberculosis) type. 20 However, Mycobacterium avium causes most mycobacterial disease i n swine i n the U.S. t o d a y . - A d d i t i o n a l evidence implies that M. avium serotypes 1 and 2, which also cause disease in chickens, are the organisms most frequently associated with tuberculous lesions in s w i n e , ^ a l t h o u g h many myco bacteria in the M. avium complex are also involved.^' Other species of mycobacteria less frequently isolated in connection with swine tuberculosis include M. bovis, M. tuberculosis, M. scrofulaceum, M. xenopi, M. kansasii, M. microti, M. ague, and M. chelonei. 20 ' 36 ' 37, 62, 102, 105, 108 Corynebacterium equi has also been isolated from lymph nodes of swine with tuberculos+s^Tfce—'lesions. 2 ^' The organism produces localized lesions and cannot be easily differen tiated from tuberculous processes macroscopically or histologically. 4 21 ' 43 ' 108 It has been reported that Corynebacterium equi is often found in association with tubercle bacilli in swine J' ^ Other workers have reported that it occurs with equal frequency in normal and M. avium-infected lymph nodes. 20 Although the relationship between £. equi and M. avium and the possible role of equi in producing tuberculosis-like lesions in swine needs further investiga tion, its effect on occurrence of the disease is probably minimal. Taxonomy Koch's report of the tubercle bacillus in 1882 was-followed in 1889 by Rivolta's description of the avian type bacillus, and in 1898 by Theobold Smith's differentiation of the bovine type bacillus. 20 However, in the 100 years since, the taxonomy of myco bacteria has become more awkward and complex. This is especially true for organisms now included in the M. avium complex. Schaefer is the person considered instrumental in taxonomy of what is now the M. avium complex. In 1935, he began his study of the serological properties of the "atypical" mycobacteria.^ He found that all avian strains were of 2 serotypes, characterized by their reactions with type-specific antisera. He also found that other "atypical" mycobacteria (Battey type) could also be typed serologically. 117 M. intracellular and M. scrofulaceum were named as species separate from M. avium, although biochemical differences among these 3 are so minimal that they cannot be reliably differen tiated by this means. Almost 20 serotypes have been established among cultures identified as M. intracellular, and several among 5 those identified as M. scrofulaceum.^' ^ In spite of objections by Runyon, who believed M. intraQC cellulare should remain a distinct species, now been grouped into the M. avium complex. these organisms have The problem has been resolved somewhat by the new scheme proposed by Wolinsky and Schaefer in 1973.^ Individual species names are dropped and the 3 are grouped together in the "Mycobacterium avium complex." Twenty numbers are reserved in the scheme after the former M. intracellular group for possible new types related to this group or to M. avium, and it is believed this new scheme will be a great aid in the study of these organisms. Epidemiology Because most swine tuberculosis in the United States is of the avian type, and appears to be most prevalent in those geographic regions where tuberculosis in chickens is greatest, it might be assumed that infected chickens are the source o f this d i s e a s e . ^ H o w ever, it should be kept in mind that this data comes from meat inspec tion records which may be misleading. Swine are often transported to abattoirs at great distance from their origin and, in addition, diagnosis is made on the basis of the macroscopic appearance of lesions. Some liesions may not be macroscopically visible and, therefore, some infections are missed. Also, there are reports of tuberculosis in hogs in areas where there is no association with chickens. Although tuberculous chickens may be the primary source of the disease in swine, it seems very probable that other aspects of the swine 108 6 environment also have a role in the origin and transmission of the disease. The opportunity for transmission of tuberculosis among tuberculous poultry, cattle, and swine has been documented by many w o r k e r s . ^ B o v i n e t u b e r c l e b a c i l l i a r e n o t a common cause of tuberculosis in swine in areas where the disease in cattle is controlled. 14 Where tuberculosis does occur in cattle, the infection may be transmitted to swine by feeding of unpasteurized milk and dairy by-products.^ Also, feces of tuberculous cattle may contain viable tubercle bacilli, which makes the maintenance of swine and cattle in common feedlots impractical.^ Tuberculosis in chickens is found in the pectoral muscles, eggs, and reproductive organs.^ 5 The feeding of offal of tuberculous chickens, and the raising of swine in areas previously occupied by tuberculous chickens have been shown to be of great significance in the transmission of avian type tuberculosis. 14 ' 23 Schalk and co workers found viable and pathogenic avian tubercle bacilli in the soil and litter of a chicken coop after 4 years with no chickens. 56 They concluded that soil contaminated by feces of tuberculosis fowl is one of the most important sources of infection for swine, and recom mended that young birds be reared on clean ground and that all fowl more than 1 year old be disposed of regularly. The transmission of tuberculosis from pig-to-pig has been attributed to ingestion of contaminated feces, 56 intrauterine infec tion, and aerosol infection from pigs showing pulmonary lesions.® 7 However, work being done in our laboratory indicates that fecal shedding of the organism does not occur, and that transmission from pig-to-pig may be rare. Human tubercle bacilli have been isolated from lesions in O swine. People known to have active TB should not be permitted to have contact with domestic animals. Soil, water, and house-dust have also been found to be possible sources of mycobacterial infection. 28 ' 56 ' 84 Sawdust is often used as bedding for swine and has recently been shown to be a source of mycobacterial infection for swine. 81 , 85, 95, 96 jn g stuc jy ^ Beerwerth and Popp, M. avium complex organisms were isolated from more than 3% of sawdust and wood shavings samples examined. Reznikov and Leggo found sawdust to be an especially favorable medium for the multiplication of these organisms due to the relatively high temperatures that develop in the sawdust piles. 84 Be cause swine often ingest sawdust, this reservoir could be an important source of infection and could account for many of the cases of M. avium complex disease in swine. Wildlife is another possible source of the infection for pigs. 6 34 38 Many wild birds have been shown to be infected. ' ' As early as 1940, Hignett and McKenzie reported the occurrence of avian tuberculosis in starlings to be 1.3 - 4.8%. 34 They suggested the possibility of the starling acting as the vehicle of infection in the spread of avian tuberculosis. Tubercle bacilli could have been introduced into the swine herd by these birds. Other wild birds found infected with members of the M. avium complex include a trumpeter swan, crane, 103 and a white-headed tree duck. 42 a sandhill This emphasizes the impor tance of adequate quarantine and test procedures to detect diseased birds before importation. There has been an increased interest in tuberculosis in cap tive exotic species following recent reports of the disease in ani mals in zoos, primate centers, animal colonies at universities, and animal and game parks. The importance of these occurrences of tubercu losis is emphasized due to the public health hazard, the economic losses, and the difficulty of replacing rare and endangered exotic animals. Thoen et al. summarize results of mycobacteria ogic examina tions conducted on 474 specimens submitted from exotic animals sus pected of having tuberculosis.^^ The widespread occurrence of tuber culosis in exotic animals maintained in captivity emphasizes the importance of these infections. The exact source of tuberculosis for swine is not always known. The organisms may be present in many areas of the swine environment or originate from only 1 source common to all animals. Many workers agree that the key lies in more accurate and reliable procedures for detection of M. avium. Diagnosis Clinical signs are of limited value in the diagnosis of M. avium infection in swine. Animals with progressive disease very sel dom show symptoms of an infectious disease. If symptoms are present, they are not sufficiently characteristic to establish a diagnosis of 9 tuberculosis. In cases of rapidly disseminating disease, a circum stance which is not characteristic of M. avium infection, there may be clinical evidence of infection including an elevation in body temperature, anorexia, and weight loss.^' ^ Tuberculous lesions in swine are usually limited to the lymph nodes of the pharyngeal and cervical regions and of the mesentery. 108 19 ' However, the lesions may be found to involve such organs as liver, lung, spleen, and kidneys in widely disseminated or generalized cases. 14 ' 17' »• 109 As stated previously, tuberculosis in swine may be caused by M. bovis, M. tuberculosis, or M. avium. Differentiating between porcine lymphadenitis caused by M. avium and that caused by M. tuberculosis or M. bovis on the basis of the gross lesions they produce is difficult. Thoen and Karl son described some gross features characteristic of each. 109 In avian tuberculosis, the lymph nodes are enlarged and firm with no discrete purulent foci; there may be 1 or more soft caseous areas with indistinct borders. Calcification is seldom seen. The cut surface of a lesion has a neoplastic appearance with a few caseous foci. There may be diffuse fibrosis but no encapsulation; the granu lomas are not easily enucleated. Mammalian lesions, on the other hand, are well encapsulated and easy to separate from surrounding tissue, calcification is prominent, and individual foci are discrete and caseous. Microscopically, avian tubercle lesions demonstrate diffuse proliferation of epithelioid cells and giant cells. There may be some necrosis but calcification is seldom seen and only in older lesions. There is a proliferation of connective tissue elements, but there is little tendency to form a well-defined wall of fibrosis. Mammalian lesions become encapsulated by a well-defined zone of connective tissue. Early caseation and marked calcification are also non+ 6» 1°6> prominent. 109 It is generally agreed that it is impossible to identify the causative agent on the basis of gross or microscopic appearance. This is probably due to the fact that there is a great similarity between localized tuberculous lesions and those associated with C^. equi and other bacteria. Furthermore, it may be difficult to differentiate among granulomatous lesions due to M. avium infection, parasitic nodules, and neoplasms. 109 A definitive diagnosis can only be made on the basis of bacteridogic procedures designed for isolation, identifi cation and typing of mycobacteria. However, there is 1 drawback. It is well known that tubercles can resolve into nodules of fibrous tissue failing to yield tubercle bacilli. 83 Cultures should be incubated at 41°C in an atmosphere of 5-10% COg; room temperature and 37°C without COg are also adequate. Cultures should be examined daily for the first week and then weekly for at least 8 weeks. Usually growth occurs in 14-21 days on solid media, but it may take up to 5 weeks. Colonies are round, smooth and glistening with a light buff-yellow color. white and translucent. On Middlebrook 7H10 colonies are flat, Morphologically, M. avium is a short, acid-fast rod in lesions, but in cultures it may develop as acid-fast branching filaments. 20, 41' 53 11 Routine biochemical tests on Mycobacterium spp. include niacin production, hydrolysis of Tween 80, nitrate reduction, and tellurite reduction. M. avium is negative for all the above. 20 In the outer layer of their cell walls mycobacteria contain a substance known as Wax D that is responsible for the relative difficulty of staining of these organisms by conventional methods. This problem is overcome by using the classical acid-fast stain. 41 With this stain, mycobacteria stain cranberry red. Two fluorescence .techniques are currently employed in myco bacterial staining: auramine O-acridine orange (AOAO) and the indirect fluorescent antibody technique (IFAT). 29 ' 39 ' 72 The former is more simple and requires fewer and less costly reagents, but the latter may aid in speciating an isolate. A special technique has been developed for detection of mycobacteria by electron microscopy. 32 The tuberculin test, used on a herd basis, seems to be a use ful tool in the diagnosis of tuberculosis in swine. Tuberculins are mixtures of soluble components of mycobacteria grown in liquid media. Robert Koch, in an effort to find a treatment for tuberculosis developed old tuberculin, a concentrate of the broth culture filtrate. It produced a marked reaction when injected intradermally into people with tuberculosis. Old tuberculin was later shown to be unsuitable for treatment, but its usefulness as a diagnostic tool was quickly realized. Currently, a purified protein derivative (PPD) of myco bacteria is used. These reagents are fractions of the culture fil- trate prepared by various methods of precipitation. 3 PPD reagents 12 have been prepared from M. tuberculosis, M. avium, M. fortuitum, M. scrofulaceum, M. marinum, and M. kansasii. PPD is now produced from mycobacteria grown in synthetic liquid medium rather than in broth. 3 This procedure yields a very pure product, free from nonmycobacterial protein. The test is usually performed by injecting 0.1 cc of tuberculin intradermally into either the ear or vulva, and results are read 48 and 72 hours after administration. 65 The size, shape, and consistency of the tissue response do not reflect the degree of infection. Unfortu nately, reports indicate that results from tuberculin skin tests in pigs may be unreliable. As early as 1952, Luke reported that there was a large percentage of error due to non-specific sensitivity or to residual sensitivity from healed lesions.^ Animals with negative skin test reactions had lesions at slaughter which Luke ascribed to the ability of the pig to overcome and apparently sterilize existing lesions. There still exists some controversy over the reliability and reproducibility of the tuberculin skin test. A peripheral blood lymphocyte stimulation test has been studied and compared with the tuberculin skin test. 5 ^' ^ 06 Results indicate that in vitro lymphocyte immunostimulation may be of considerable value in detecting tuberculosis in animals in which skin testing has been used previously.^®' ^ More work needs to be done on this test, especially a comparison between the 2 tests using naturally infected pigs. Lastly, the enzyme-linked immunosorbent assay (ELISA) has recently been described for detecting antibodies in swine infected with 13 M. avium.^ Positive ELISA reactions were seen in experimentally and naturally infected pigs. It is a rapid, automated test, which may be of value on a herd basis. Pathogenesis The mechanism by which mycobacteria produce disease in man and animals is relatively unknown. It is not known which properties of the bacteria are responsible for virulence, and the fact that the pathogenicity varies within different species of animals makes this matter even more complex. Different hosts seem to be susceptible to different types of mycobacteria. Rabbits, cattle, and cats are susceptible to M. bovis, but M. tuberculosis causes little problem in these animals. Man and guinea pig are very susceptible to M. tuberculosis, however, the rat is resistant to this species.^' ^ In man, differences in age have been reported to affect the incidence of M. tuberculosis infection. The disease is found to be more frequent in the very young and in the elderly, perhaps due to the occasional inadequate immune status of these age groups. 139 Pregnant women seem to be more susceptible to tuberculosis for the same reason. Other studies have shown that the body temperature of the host may also play a role in susceptibility. Chickens, whose body temper ature is 42°C, are susceptible to infection with M. avium which grows well in vitro at 43°C. On the other hand, chickens are resistant to M. bovis and M. tuberculosis which require growth temperatures of 37°C. It has been known for some time that immunity to infection by facultative intracellular parasites, such as mycobacteria, is not mediated by an antibody. 118 This fact is supported by the fact that in either the natural or experimental disease, there is no relation ship between the amount of antibody formed and immunity to tuberculosis. 82 Experimentally, workers have been unable to transfer immunity from immunized animals to nonimmunized animals by injecting serum, 82 but have now shown that immunity can be transferred by using lymphoid cells taken from infected animals. 98 ' 99 About 2-4 weeks following infection with tubercle bacilli, the host's cellmediated immune response attempts to destroy the organism. The macro phage seems to be the most important cell type involved in this destruction process. is engulfed. 15 The bacillus attaches to the macrophage, and This results in the formation of a phagosome. These phagosomes then fuse with lysosomes containing hydrolytic enzymes. This fusion results in the formation of phagolysosomes in which hydrolytic enzymes destroy the microorganism. are resistant to intracellular destruction. However, mycobacteria In fact, the tubercle bacilli multiplies in and will usually kill the macrophage. It is not certain how mycobacteria accomplish this feat, but different theories exist. The tubercle bacilli may use the enzymes present in the phagolysosome, or it may block fusion of the phagosome and lysosome. 26 It is generally agreed that the resistance of mycobacteria to lysozyme is due to the high lipid content of the cell wall. One of the outstanding features of the host-tubercle relation ship is the formation of granulomas. This granulomatous inflammation first becomes evident in 2-4 weeks, after cellular immunity develops. 116 Macrophages accumulate at the site of infection, and enlarge following ingestion of bacilli. Macrophages are modified, lose their phago cytic ability, and are termed "epithelioid" cells because of their resemblance to epithelial cells. Epithelioid-cell formation is accompanied by a tendency for the cells to be arranged in groups rather than in diffuse sheets. Frequently, when macrophages encounter insoluble material or organisms causing chronic inflammation, they coalesce to form giant cells. Therefore, in a tuberculoid reaction, in addition to epithelioid cells, a considerable number of giant cells are also present. Surrounding the mass of epithelioid cells, there is a diffuse zone of lymphocytes with a few plasma cells and fibrocytes. Within 2 weeks, caseation necrosis takes place, and the tissue forms a dry, firm, coagulated mass. This is thought to be caused by the development of hypersensitivity to the bacilli. The end result is a fully formed tubercle follicle, consisting of a central mass of caseation surrounded by epithelioid cells and giant cells. This, in turn, is surrounded by a zone of round cells. With swine tuberculosis, these tubercles can be found most commonly in cervical or mesenteric lymph nodes. As previously stated, the properties which cause mycobacteria to be pathogenic are poorly understood. A factor noted early which may relate to pathogenesis of tubercle bacilli is the formation of cords by the organisms in liquid medium. It is thought that the cell wall and its lipid components, in particular a lipid identified as trehalose-6, 6'-dimycolate, 55 ' 73 " are responsible for this phenomenon. Cord factor can be extracted, and is known to inhibit the migration of lymphocytes.^ Experimentally, it was noted that some animals, inocu lated with extracted bacilli, developed disease more slowly than those inoculated with unextracted bacilli. Further studies showed that the level of succinic dehydrogenase in the animal decreased with an increase in the severity of tuberculosis.^ Cord factor apparently inhibits host production and/or secretion of succinic dehydrogenase and, therefore, the host is less able to prevent, or more likely control, advancement of the infection. 45 Cord factor has also been shown to cause swelling and lysis of mouse liver mitochondria. 47 The effect of cord factor on other organelles has also been studied. It has been found to induce sepa ration of the ribosomes from the rough endoplasmic reticulum of liver cells in mice. 24 The nature of these actions is not well understood. Other compounds isolated from virulent mycobacteria are sulfolipids. sulfolipids. 27 In fact, many species are known to have common Sulfatides have been shown to prevent fusion of phago- somes and lysosomes. 47 This would allow the bacilli another means, aside from cord factor, of escaping and/or prolonging digestion by lysosomal enzymes. affect mitochondria. These sulfolipids, like cord factor, are known to In fact, it appears the 2 compounds may act synergistically, as sulfolipid was found to drastically increase the toxicity of cord factor.^ During phagocytosis, there is an increased production of superoxide by the host tissue, another attempt at stopping infection. 17 An iron-containing superoxide dismutase has been isolated from M. tuberculosis, and has been found to protect the organism from the toxic effect of superoxide. 52 ' With M. avium, colony morphology is correlated with virulence. 113 Those colonies which are flat, transparent, and colorless are usually more virulent than those that are dome-shaped, smooth, and opaque. These "virulent" colonies can also be identified by their growth requirement for palmitic acid; nonpathogenic M. avium has no such requirement. 64 M. avium infection in swine is primarily a disease of the lymph nodes associated with the gastrointestinal tract, more specifi cally the small intestine. The following is a brief discussion of the anatomy and physiology of the small intestine with an emphasis on those structures involved in the uptake and transport of macromolecules from the gut. The small intestine has 4 main functions: secretion, motility, digestion, and absorption. 87 ' 94 Here, we are mostly concerned with absorption or the mechanisms by which molecules transverse the intes tinal wall. Through an understanding of these mechanisms involved in absorption, a better understanding of the potential mechanisms by which M. avium may travel from the intestinal lumen to the regional lymph nodes may be obtained. The wall of the small intestine is composed of 4 layers: the mucosa, submucosa, muscularis externa, and serosa. The mucosa is composed of an epithelial cell layer with its filamentous basement membrane. The basement membrane is a lamina propria containing blood vessels, lymphatics, smooth muscle cells, nerve fibers, plasma cells, lymphocytes, fibroblasts, eosinophils, macrophages, reticular cells, mast cells, collagen, and reticular fibrils and is separated from the submucosa by the muscularis mucosa. The submucosa contains larger blood vessels, lymphatics, more connective tissue, nerves and ganglia, and more lymphoid elements. The muscularis externa is divided into an inner circular layer and an outer longitudinal layer of smooth muscle, with the myenteric plexus between the two.®''' ^ The small intestine is characterized by 3 structures which increase its surface area and aid in absorption. circumferential folds of mucosa and submucosa. First, there are They are responsible for the "feathery" appearance of the small intestine. Second, the villi are projections of mucosa which are usually fingerlike. The individual cells of the villi possess filamentous microvilli, which in turn are coated with a more fine filamentous structure. Lastly, the mucosal crypts lie between the villi and extend to the muscularis mucosa. The surfaces of the villi and the linings of the crypts are covered by a layer of columnar cells. 87 ' 94 The microvillus plasma membrane is covered with an adherent, filamentous structure called the glycocalyx. It is composed of sulfated, weakly acidic mucopolysaccharide which appears to be contin uously synthesized by the Golgi apparatus of the epithelial cell, especially at the villous tips. The plasma membrane and its asso- ciated glycocalyx constitute a digestive-absorptive unit. 87 ' 94 The process of absorption is initiated in the lumen of the small intestine and is completed by the specialized absorptive cells, the plasma membrane of which maintains composition and electrical charge between the lumen and inside the cell by regulating the rate at which molecules enter the cell. The difference between the function of this membrane at the cell's apical and basal surfaces results in a net movement of molecules through the cell or absorption. 77 ' 94 Because a main function of the small intestine is the absorption of lipids, and M. avium has a high lipid content, a special interest was taken in the mechanism by which lipids are absorbed. Fats are continuously broken down and emulsified into very small complexes called chylomicrons. These enter the lacteals by reverse pinocytosis, and electron microscopy has demonstrated that pinocytic vesicles appear to occupy approximately 15% of the endothelial cell cytoplasm. 87 ' 94 The GI tract of mammals is complete with immunologically competent tissue. It is capable not only of experiencing but also of mounting an immune response to antigenic stimulation. Two of the most important components of this system are the Peyer's patches and specialized cells, the microfold or M cells. The Peyer's patches lie in the lamina propria and submucosa throughout the small intestine, usually on the antimesenteric wall. They occur more frequently in more distal areas of the intestinal tract, but some are always found in the duodenum. Light microscopy has shown that regardless of how many follicles a patch contains, each 20 patch is only one follicle thick, thus preserving the close relationship between the follicle and the overlying epithelium. 77 ' 79 This tissue is also commonly called gut-associated lymphoid tissue or GALT. 77 The surfaces of Peyer's patches are characterized by specialized epithelium which differs from that of adjacent areas and possesses features which enhance trapping and uptake of antigenic and 8 77-79 particulate material. ' M cells appear responsible for antigen transport within the 8 75 79 88 epithelium. ' ' ' These variably surfaced cells with their scattered, short protuberances are interspersed among the ordinary epithelial cells. They form cytoplasmic bridges between ordinary epithelial cells, and separate the lumen from underlying nests of lymphoid cells. The reticulum formed by M cells allows cells from the lymphoid series (above) to approach the gut lumen very closely without being lost in the lumen. At the same time, the M cells main tain tight junctions with adjacent epithelial cells so that the integrity of the gut epithelium is preserved. 77 M cells transport antigens to the lymphocytes through a tubulovesicular system, repre sented by pits in the luminal cell surface. The underlying lymphoid cells then migrate into mucosal nodules, or are carried away by afferent lymphatics to mesenteric lymph nodes. In a study by Owens and Jones, they concluded that 1) the lymphoid cells contained within M cell pockets could be responding there to antigenic material trans ported from the intestinal lumen via the M cells, or 2) M cells may couple IgA to secretory components and transport the immunoglobulin complex into the lumen. 77 All of these lymphoid "structures" maintain a lymphoepithelial relationship which is similar to that of the thymus and the avian bursa of Fabricius. The former is responsible for T cell immunity and the latter, in avian species at least, regulates the development of B cell immunity. Most immunoglobulin-containing cells in the small intestine are mature plasma cells, although some immunoglobulins are found within immature plasma cells and lymphocytes. It is well established that IgA is the predominant immunoglobulin in the lymphoid cells and secretions of the small intestine. IgM, IgE, and IgD are 1 114 also present. ' Secretory IgA has been demonstrated to possess antiviral and antibacterial activity as well as the properties of isohemagglutinms. 114 Aside from these immunospecific mechanisms, any bacteria colonizing the intestinal tract would have to contend with the non specific resistance mechanisms functioning in the tract. This would require bypassing the low pH of the stomach, and the peristalsis and villous action in the small intestine. They would have to resist the toxicity of conjugated or unconjugated bile acids. However, those organisms sensitive to bile acids may avoid this by taking up residence in the upper small intestine, where the concentration of bile is low. 77 Mucin is another barrier against microbial invasion in the small intestine. Mucin traps particles, such as bacteria, and then clears them away through the villous action and peristalsis in the stomach and intestines. overcome this. However, certain bacteria are known to In fact, they may colonize mucins and use them as sources of carbon and energy.^ Indigenous microorganisms also may block, or limit, coloni zation and growth of pathogenic bacteria in the GI tract. However, this normal flora changes under conditions such as antibiotic treat ment, stress, diet, etc. Pathogens may take advantage of these circumstances, and infect only "altered" hosts.^ Although the neonatal small intestine has long been known to have the ability to absorb macromolecules, until recently it was thought that the adult mammalian gut maintained a complete barrier against such absorption. 12 ' 113 Today, there is increasing evidence that the normal adult intestine is permeable to macromolecules, not in sufficient quantities to be of nutritional importance, but in quantities that may be anti genic or biologically active. mechanism. 12 ' 113 The process employs an endocytic Initially, there is an interaction between these large molecules within the intestinal lumen and components of the microvillus membrane of intestinal absorptive cells. When a sufficient quantity of molecules comes in contact with the cell mem brane, invagination occurs, and small vesicles are formed. This process of uptake is energy-dependent and it has been shown to be inhibited by metabolic inhibitors of both glycolysis and oxidative phosphorylation. 12 After invagination, macromolecules travel within 23 membrane-bound vesicles (phagosomes) to the supranuclear region of the cell where the vesicles fuse with lysosomes to form phagolysosomes. In these structures intracellular digestion occurs. However, small quantities of macromolecules escape breakdown and may be deposited in the intercellular space by reverse endocytosis or exocytosis. 53 These molecules or antigens may come in contact with local lymphoid tissue or may pass into the systemic circulation. Numerous studies using macromolecules such as HRP (horseradish peroxidase), PVP, insulin, and vitamin have confirmed this finding.^' 70, 89, 93, 113 The pathogenic mechanism involved in the uptake and processing of M. avium from the small intestine is not known. Through studies involving other intracellular parasites, it is believed that this process may be similar to the endocytic-type uptake discussed in relation to macromolecular uptake from the gut. 7 f\ However, after the formation of phagolysosomes, intracellular digestion does not occur. Instead, tubercle bacilli reside and multiply within the involved macrophages. By escaping breakdown, bacilli may be deposited in the intercellular space by exocytosis, and come in contact with local lymphoid tissue. At this time, the cellular immune response of the host, and the corresponding granulomatogenesis, would be initiated to form the typical lesions found upon postmortem examination and slaughter. If M. avium does penetrate into intestinal epithelial cells by a phagocytic process, the various events in this process may be elucidated by studying them in vitro using cultured intestinal epithelial cells. 1) The objectives of this investigation are: To determine the maximum rate of infection of HIE cells by M. avium serotype 2. 2) To evaluate any differences in the interaction of pathogenic versus saprophytic mycobacteria with HIE cells. 3) To assess the properties of M. avium that may be responsible for its invasiveness. 4) To determine what role, if any, the host epithelial cell plays in uptake of M. avium. CHAPTER 2 MATERIALS AND METHODS Experimental Design Each assay consisted of Henle intestinal epithelial (HIE) cells grown to confluency in cluster 6 well tissue culture dishes. 3 Each well was inoculated with 0.25 mg (wet weight) of the appropriate organism suspended in minimum essential medium (MEM).' 3 Following incu bation, plates were washed in PBS, stained, and examined microscopi cally under oil immersion (100X). For all studies, 5 wells on each plate were used as treatment groups and the sixth as the control. randomly assigned to treatment groups. Individual plates and wells were 59 Thirty cells per well were counted at random, and the number of bacteria associated with each cell recorded. Each assay was performed 3 times and the results were averaged. Bacterial Cultures Cultures of M. avium serotype 2 were obtained from Dr. C. 0. Thoen at Iowa State University. Those of serotype 8 and serotype 10 originated from pigs with mycobacterial lymphadenitis in Arizona, and cultures of M. phlei and M. smegmatis from the mycobacterial collection a. Bellco. b. Gibco. Vineland, New Jersey. Grand Island, N.Y. 25 at the Trudeau Institute, Saranac Lake, NY. Stock solutions of each organism were prepared and stored at 0°C for future use to avoid an excessive number of passages from the original slants. Throughout this entire study, no bacterial culture exceeded 4 passages from the original slant. c d Tubes of Dubos broth containing 1.0% horse serum were inoculated with the appropriate culture, and incubated at 37°C until a density equivalent to a McFarland Tube 1 was reached. The suspensions were aseptically transferred to sterile vials containing 2.0 ml final volume and frozen at 0°C. Seven days prior to assay, the vials were thawed in a 37°C water bath. One ml of the stock culture was inoculated into 8.0 ml of Dubos broth containing 1.0% horse serum. Following 7 days incubation at 37°C, each bacterial culture was centrifuged at 2000 rpm for 20 minutes, and resuspended in 40.0 ml of MEM. Two mis of this suspension were overlayed on each well. This resulted in approximately 0.25 mg (wet weight) of bacteria per well. Cell Cultures Henle 407 human intestinal epithelial (HIE) cells e were cultured in MEM containing 10.0% fetal calf serun/ (FCS), 200 IU of c. Difco. Detroit, Michigan. d. Gibco. Grand Island, N.Y. e. Maryland. f. American Type Culture Collection (CCL 2). Gibco. Grand Island, N.Y. Rockville. penicillin per ml, 200 ug of streptomycin per ml, and 5 ug of 9 n fungizone per ml in 25.0 cm tissue culture flasks. All media were filter-sterilized using a 0.20 y cellulose-acetate filter. Once a monolayer was established, cultures were either divided 1:3 into new flasks to facilitate new growth, or transferred 1:1 into cluster 6 well tissue culture plates for assay. The procedure for passage of cells is outlined below. 1. Flasks containing monolayers were washed twice with MEM without FCS. 2. 1.0 ml of 0.25 percent trypsin -0.10 percent EDTA solution was added to each flask. 3. Flasks were incubated at 37°C with 5% CO2 until gentle tapping detached the monolayer from the vessel (about 2 minutes). 4. A 2.0 ml solution of MEM containing 10% FCS was added to each flask to neutralize the action of trypsin and to wash the cells from the surface of the flask. 5. The contents of each flask were dispensed into 15.0 ml centrifuge tubes, and centrifuged at 800 rpm for 10 minutes. 6. The supernatant fluid was discarded, and the pellet was resuspended in MEM containing 10% FCS and 2.0% antibiotic. g. Bellco. It was at this point that a bifurcation Vineland, New Jersey. 28 in the procedure occurred dependent upon whether the passage was into another flask or into a 6-well plate for assay. FLASK 7a. 8a. The pellet was resus- PLATE 7b The pellet was resus- pended in 1.5 ml of MEM pended in 12.0 ml MEM containing 10% FCS and containing 10% FCS and 2% antibiotic. 2% antibiotic. 0.5 ml of this solution 8b. A sterile coverslip was dispensed into each was placed in each well 2 of three 25.0 cm flasks. of the tissue culture plate for assays using the I FAT. 9a. The total volume was 9b 2.0 ml of the bacterial adjusted in each flask to suspension in (7b) were 5.0 ml with MEM contain added to each well. ing 10% FCS and 2% anti biotic. 10a. Flasks were incubated at 10b. Plates were incubated 37 C with 5% COg until at 37 C until conflu confluent. ent. (4 days) (3-4 days) Antiserum Preparation Antiserum against M. avi um serotype 2 was prepared for use with the indirect fluorescent antibody stain. Antisera against serotypes 8 and 10 were previously prepared by Dr. J. G. Songer, and were available for testing. Antisera against M. phlei and M. smegmatis were not available, and alternative staining techniques were employed for assays involving these bacteria. The preparation of hyperimmune serum against serotype 2, the immunization of rabbits, and the collection of serum was as follows: L 1. Tubes of Dubos broth containing OADC J and Tween 80 were inoculated with M. avium serotype 2, and incubated at 37°C for 14 days. 2. One ml of this suspension was transferred to each of L several plates of Middlebrook 7H10 Agar containing OADC enrichment and 4.1 mg of sodium pyruvate per ml of medium. Plates were incubated at 37°C for 14 days. 3. Cultures were checked daily for contamination. If pure, phenolized phosphate buffered saline (PPBS) was added to each plate, and the cells harvested. 4. Cells remained in PPBS, at a density equivalent to a McFarland III Standard, for 5 days. 5. Phenolized cell suspensions were centrifuged and the pellet was resuspended in 2.0 ml of PBS. This was used to prepare a cell suspension of 0.4 optical density at 525 nm in PBS. h. Difco. Detroit, Michigan. i. Sigma. St. Louis, Missouri. 30 6. The marginal ear vein of New Zealand White rabbits was injected with 1.0 ml of the final suspension on days 0, 4, 8, and 12. 7. Two mis of the same suspension were injected into the ear vein on days 16, 20, 24, and 28. 8. Rabbits were exsanguinated on day 35. The blood was allowed to clot and then centrifuged. 9. The serum was collected and stored at -20°C until needed. Method of Assay Kinetics Studies Kinetic studies were performed on M. aviurn serotypes 2, 8, and 10, M. phlei, and M. smegmatis. Each assay required 6 tissue culture plates and 60 ml of the appropriate bacterial culture. Each well was washed twice with MEM and 2.0 ml of the bacterial suspension were overlaid onto the monolayer of each of 5 wells. tained MEM only, and served as a control. The sixth well con Plates were incubated at 37°C for 30, 60, 120, 180, 240, or 300 minutes, after which the bacterial suspension was aspirated, and each well was washed 4 times with PBS. Cells were then fixed for 30 minutes, or processed without fixation, depending upon the staining technique to be used. Assays Involving Phagocytic Inhibiting Reagents These studies involved M. avium serotype 2 only. The proce dures for preparing HIE cell monolayers and bacterial cultures were the same as previously described, except covers!ips were not used. The reagents used included cytochalasin B 1 at concentrations of 0.05, 0.10, 0.50, 1.0, 2.0, and 4.0 mg/ml, dibutyryl cyclic AMP 1 ' at concen trations of 1.0, 2.0, 4.0, and 10.0 mM, iodoacetic acid 1 at concentra tions of 0.01, 0.03, 0.05, .10, .20, and ,30 mM and 2,4-dinitrophenol 1 at concentrations of 0.001, 0.01, 0.1, 1.0, and 2.0 mM. All reagents were diluted in MEM except cytochalasin B which was prepared as a 1.0 mg/ml stock solution in dimethyl sulfoxide 1 and then diluted in MEM. Two different infection protocols were used in these assays. In Procedure A, 2.0 ml of the appropriate concentration of reagent were applied to each well, and the plates were incubated at 37°C for 3 hours. Following incubation, monolayers were washed 4 times 'in MEM and infected with 2.0 ml of the appropriate bacterial suspension. After an additional incubation period of 3 hours at 37°C, plates were washed 4 times in PBS and fixed with formalin for 30 minutes. In Procedure B, equal volumes of the bacterial suspension and reagents were incubated together at 37°C for 3 hours. This suspension was used to infect monolayers by overlaying each well with 2.0 ml of the appro priate solution and incubating an additional 3 hours at 37°C. Each well was then washed 4 times in PBS and fixed with formalin for 30 minutes. All plates were stained with the acid-fast stain. One plate was run for each reagent per replication except in those instances where there were more dilutions than wells on each plate, and then it was necessary to run 2 plates for that reagent. Each assay incorpor ated 1 monolayer not subject to chemical treatment to serve as a j. Sigma. St. Louis, Missouri. 32 control. Studies Involving "Altered" Bacteria These studies involved M. avium serotype 2 only. The procedure for preparing HIE cell monolayers and bacterial suspensions were the same as previously described. A 7 day culture of M. avium, grown in Dubos broth, was autoclaved and centrifuged at 2000 rpm for 20 minutes. The supernatant fluid was discarded and the pellet was resuspended in 40 ml of MEM. Prior to infection of monolayers with this suspension, Middlebrook 7H10 Agar plates were inoculated to determine the viability of mycobacteria after autoclaving. Two confluent, 6-well plates of HIE cells were prepared per replication. After washing each well twice with MEM, the cells from randomly selected wells were infected with 2.0 ml of the above bacterial suspension. The sixth well was overlaid with 2.0 ml of viable M. avium serotype 2 suspended in MEM, and served as the control. Following 3 hours of incubation at 37°C, the coverslips were removed from each well, labeled, and washed 4 times in PBS. The mono layers, grown on coverslips from 1 plate were fixed in methanol for 30 minutes, while those from the second plate were processed without fixing. Those experiments involving formalinized M. avium used the same protocol as above except the 7 day culture of M. avium was centrifuged and resuspended in 5% formalin for 15 minutes. Cells were washed twice in PBS before resuspension in 40 mis of MEM. To determine the effect of UV irradiation on M. avium in this system, a 7-day culture of M. avium was centrifuged at 2000 rpm for 20 minutes and resuspended in 40 ml of MEM. Four ml of the above suspension were placed in each of 6 100 x 15 mm petri dishes a shallow layer. k to form The petri dishes were exposed to UV light for 10, 20, 30, 40, 50, or 60 seconds from a distance of 60 cm. After irra diation, the contents of each petri dish were cultured on Middlebrook 7H10 Agar to check viability. Using two 6-well tissue culture plates as treatment groups, each treated bacterial solution was randomly assigned to 1 of the 6 wells per plate. were overlaid onto each monolayer. Two ml of this suspension The third plate was used as the control, and cells from each well were infected with 2.0 ml of M. avium in MEM that had not been exposed to UV irradiation. were incubated for 3 hours at 37°C. All plates Coverslips, upon which monolayers had been grown, were removed from wells, labeled, and washed 4 times in MEM. The coverslips from 1 of the treated plates were fixed in methanol for 30 minutes, while coverslips from the other treated plates were processed without fixing. Three randomly selected coverslips from the control plate were also fixed in methanol for 30 minutes while the remaining 3 were not fixed. M. avium was suspended in MEM containing various dilutions of antisera to observe the possible effect of masking surface molecules functioning as receptor sights in attachment. Serum was filtered through a 0.20 y cellulose-acetate filter before being diluted 1:16, 1:64, and 1:256 in MEM. k. Falcon. Each replication of this assay required Oxnard, California. 34 monolayers in three 6-well tissue culture plates, and three 7-day cultures of M. avium. The acid-fast stain was employed in this assay so it was not necessary to incorporate coverslips in these tissue culture plates. Bacterial cultures were centrifuged at 2000 rpm for 20 minutes before suspension in 40 ml of 1 of 3 antibody-containing solutions of MEM. infection. All monolayers were washed twice in MEM before Two ml of the appropriate bacterial suspension were added to each of 5 wells. The monolayer in the sixth well was inocu lated with M. avium suspended in MEM not containing antisera. Plates were incubated for 3 hours at 37°C, washed 4 times in MEM, and fixed with formalin for 30 minutes. The format followed in studies involving M. avium cultures suspended in milk or colostrum was essentially the same as that described above, except the FA staining technique was used. Two con fluent 6-well tissue culture plates with coverslips, and one 7-day culture of M. avium were needed per replication for each of the 2 treatments (milk or colostrum). The unpasteurized milk and colostrum were obtained from the University of Arizona Dairy from cattle not receiving antibiotic therapy, and the pH was adjusted to 7.0. A culture of M. avium was resuspended in 40 ml of milk or colostrum, and 2.0 ml of this suspension were overlaid onto each of 5 monolayers. The sixth monolayer, the control, was infected with M. avium suspended in MEM. Plates were incubated 3 hours at 37°C, after which the cover- slips, containing monolayers, were removed, labeled, and washed 4 times in MEM. The coverslips from 1 plate were fixed in methanol for 30 minutes, while those from the second plate were processed without fixing. Staining Techniques Kinyoun's Acid-Fast Stain This procedure was employed initially with cultures infected with M. avium serotype 2 to determine if there was any attachment occurring, in all assays involving M. phlei and M. smegmatis, and in those incorporating M. avium suspended in antiserum. The procedure described below begins after fixation of cultures with formalin: 1. Carbol fuchsin^ 1 Hour 2. Rinse 70 percent alcohol 2 Times 3. Rinse running tap water 5 Minutes 4. Rinse acid alcohol 3 Times 5. Rinse running tap water 8 Minutes 6. Methylene blue^ 30 Seconds 7. Rinse running tap water 10 Minutes Auramine O-Acridine Orange Stain This stain was used on all cultures involving the phagocytosis inhibiting reagents. After fixation in formalin, infected monolayers were treated as follows: 1. Auramine 0^ (0.30% solution) 2. Rinse running tap water 5 Minutes 3. Rinse acid alcohol 2 Times 1. Fisher. Fair!awn, New Jersey. 15 Minutes 36 4. Acridine orange m 5 'Minutes 5. Rinse running tap water 5 Minutes Indirect Fluorescent-Antibody Stain Immunofluorescent techniques similar to those described by 49 Kihistrom and modified to incorporate a counterstain were used to differentiate intracellular and cell surface adherent bacteria in kinetic studies involving M. avium serotype 2, serotype 8, and sero type 10. Assays incorporating autoclaved, ultraviolet exposed, formalin treated, and milk or colostrum coated M. avium serotype 2 were also evaluated using this procedure. Monolayers, grown on cover- slips, were either fixed in methanol or processed without fixation according to the following protocol: 1. Monolayers were incubated with a 1:64 dilution of rabbit anti-M. avium serotype 2 antibody for 20 minutes at 37°C. A dilution of 1:160 was used for serotype 8 and a dilution of 1:320 was used for serotype 10. 2. Cells were washed 4 times in PBS. 3. Monolayers were incubated with 1:32 dilution of fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit 75 immuno globulin 11 for 20 minutes at 37°C. 4. Cells were washed 4 times in PBS. 5. A counterstain, consisting of 0.125 percent Evans Blue m m. Sigma. St. Louis, Missouri. n. Mi 1es. Elkhard, Indiana. 37 diluted in PBS, was applied to each coverslip for 10 minutes. 6. Cells were washed twice in PBS and mounted in 60% glycerol. Microscopy Light Those cultures stained with Kinyoun's acid-fast stain were examined under oil immersion using the 100 x objective. Fluorescence Cultures stained with the Auramine 0-acridine orange stain and those involving the indirect fluorescent antibody stain were examined by incident light fluorescence microscopy at 100X (oil immersion) using a Leitz photomicroscope with exciter filter II and a 400 nm barrier filter. Statistical Analysis The data were distributed according to a Poisson distribution, with numerous small integer and zero counts. because data were found to be highly skewed, a For this reason, and 1/ X + .5 transformation was used on most bacterial counts. Two statistical computer programs, compiled by Kelley, used for data analysis. 48 were The first included an analysis of variance and tests of significance between treatment groups (a = .05) were per formed with the F-test. A second program was devised to find the mean 38 count for each treatment group per assay. Therefore, each data point consisted of the average of 3 means corresponding to the 3 performances of each treatment. When the percent of intracellular bacteria was needed, the means for fixed and unfixed monolayers were employed in the following formula: Mean Number of Bacteria/Fixed HIE Cell Mean Number of Bacteria/Unfixed HIE Cell Mean Number of Bacteria/Fixed HIE Cell x 100 All data from those assays involving reagents to inhibit phago cytosis were evaluated without transformation. into 2 groups: infected and noninfected. ciated bacteria were defined as infected. The cells were arranged Cells with 1 or more asso This was done for each con centration of each reagent, and because each assay was performed 3 times, an average of the 3 was used in the following formula to give the percentage of HIE cells infected per monolayer: Mean Number of Cells Infected Mean Number of Cells Infected + Mean Number of Cells Noninfected x 100 Tests of significance were conducted using the Newman-Keuls mean separa tion test and a 95% confidence level. In some cases, it was necessary to look individually at fixed and unfixed HIE cell monolayers. Bacteria could be considered intra cellular if the 2 preparations were found to stain differently, and extracellular if preparations stained alike. Using raw data, each HIE cell counted was placed in 1 of 7 groups depending upon the number of bacteria which were found in association with it. Groups included 0, 1-10, 11-20, 21-30, 31-40, 41-50, arid 51-60 associated bacteria. The distributions for fixed and unfixed monolayers were then graphed for comparison. CHAPTER 3 RESULTS AND DISCUSSION Kinetics Studies Primary assays involving M. avium serotype 2 were conducted with the intention of establishing the amount of time required for maximum infection of cultured epithelial cells. Data indicate that maximum cellular uptake occurred after 3 hours incubation time. (Figure 1.) Because mycobacteria are intracellular parasites, the validity of the data in Figure 1 is dependent upon the assumption that cell-associated bacteria seen in acid-fast stained monolayers were in fact intracellular and not merely adhering to host cell surfaces. A 49 technique described by Kihlstrom was used to aid in distinguishing surface-associated from internal bacteria. This indirect fluorescent- antibody test is based upon the fact that immunoglobulin proteins do not cross the intact plasma membrane^ but diffuse freely into methanol-fixed cells. 92 This principle was first applied to monolayers infected with M. avium serotype 2 for time intervals of 30 minutes to 5 hours. time. Figure 2 shows the percent intracellular bacteria at each Maximum infection of epithelial cells occurred after 3 hours of incubation, and at this time, over 60% of the bacteria seen in associ ation with each epithelial cell were, in fact, intracellular. The fact that the number of intracellular bacteria per HIE cell decreased as incubation time exceeded 3 hours, may be due to 40 5.01— 4.5 4.0 O 0s a> <5 3.5 30 0J o 2.5 o CD •o a> O ~ "o 2.0 8CO < v»— o 1.5 w. a> .a E z 1.0 0.5 30 60 120 180 240 Time (minutes) Figure 1. Kinetics of the Interaction of M. avium Serotype 2 with HIE Cells. 300 I00.0|— 90.0 80.0 o 3* $ 70.0 ® 60.0 a> O 50.0 m = 40.0 a> o a c 30.0 8 a. 20.0 10.0 J 30 I 60 I 120 I 180 I 240 I 300 Time (minutes) Figure 2. Percent of M. avium Serotype 2 Shown to be Intra cellular when Associated with HIE Cells. 43 exocytosis of tubercle bacilli. 53 The kinetics of the interactions of serotypes 8 and 10 with HIE cells were studied.to determine if they differed from the supposedly more pathogenic serotypes 1 and 2. Serotype 8 exhibited maximum uptake of 42.94% after 3 hours incubation (Figure 3). 13% less than serotype 2 uptake. is even less with serotype 10. This was Figure 4 shows that cellular uptake Furthermore, this maximum uptake of only 28.60% occurred after only 2 hours incubation. The differences in kinetics of the M. avium:HIE cell interaction may partly explain the differences in rates of infection with various serotypes. Other factors, such as the relative prevalence in the environment of the different serotypes, may also play a role. Saprophytic mycobacteria, which are not causally associated with a disease process, are widespread in the environment. Two of these, M. phlei and M. smegmatis, are occasionally isolated from tuberculous lesions of swine, often with M. avium. The interaction of these 2 organisms with HIE cells were studied to compare them with pathogenic M. avium. Results are shown in Figures 5 and 6, as the mean number of bacteria associated with each HIE cell. The lack of antisera against these species of bacteria did not allow use of IFAT. Neither organism displayed a significant (P = 0.05) ability to attach to or penetrate, HIE cells. If an equivalent response occurs in vivo, it may explain the rare occurrence of these, as compared to M. avium, in porcine lymph nodes. tOO.Oi— 90.0 ^ 8Q0 & 70.0 •H « 60.0 N O 'k a> "g 50.0 m = 40.0 a> CJ o w. C 30.0 3 (3! 20.0 10.0 30 J 60 I 120 L 180 240 300 Time (minutes) Figure 3. Percent of M. avium Serotype 8 Shown to be Intracellular when Associated with HIE Cells. 100.0 90.0 8Q0 STO.0 •H « 60.0 a> S 50.0 CD = 40.0 d) o o 30.0 8 i2! 20.0 10.0 I 30 1 60 I 120 I 180 I 240 Time (minutes) Figure 4. Percent of M. avium Serotype 10 Shown to be Intracellular when Associated with HIE Cells. L 300 5.0 4.5 —. 4.0 SU 3.0 25 '§ 20 8 (O < »•— o 1.5 1.0 Q5 30 1 1 60 120 180 240 300 Time (minutes) Figure 5. Kinetics of the Interaction of M. phlei with HIE Cells. 5.0 4.5 4.0 O in „ <X> 3.5 <330 0> o 2.5 o CD "O a> •§ 20 8 U) < H- O |.5 k. a> -O E 1.0 0.5 J30 I 60 I 120 L 180 240 300 Time (minutes) Figure 6. Kinetics of the Interaction of M. smegmatis wi HIE Cells. 48 The interaction of M. aviurn serotype 2 with epithelial cells was examined in greater detail by studying separately the contributions of the HIE cells and the bacteria to the uptake process. Host Cell Effects On Uptake HIE cell monolayers were treated with 4 compounds known to inhibit the ability of phagocytic cells to engulf particles. Pre incubation of host cells with the appropriate reagent (Procedure A) versus treatment of bacterial cultures with each reagent before infecting monolayers (Procedure B) was designed to assure that the effect of each reagent was upon the host cells and not upon the bacteria. Cytochalasin B, a fungal metabolite, inhibits phagocytosis by . . 2 4 polymorphonuclear leukocytes (PMN) and macrophages. ' Many investi gators have reported the disruption of actin polymers of microfilaments in both phagocytic and nonphagocytic cells treated with cytochalasin B.2' ^ According to these authors, concentrations of 1-3 mg cytochalasin B/ml disrupt microfilaments and suppress cell phagocytosis. Results in Table 1 indicate significant inhibition of phagocytosis at a concentration of 1.0 mg cytochalasin B/ml, regardless of which procedure was applied. This suggests that infection of epithelial cells in vitro is dependent upon functional host cell micro filaments. Dibutyryl cAMP inhibits particle ingestion by PMN and mouse Table 1. Effect of Cytochalasin B on Interaction of M. avium Serotype 2 with HIE Cells. PROCEDURE Concentration (Uq/ml) .AA x % 4.0 16 SO 2.0 1.0 0.5 0.1 0.05 0 22.33 25.33 39.33 45.33 56.33 59.66 60 60 60 60 60 So~ 4.0 2.0 1.0 0.5 0.1 0.05 0 6.66 16.66 21.66 3'».66 40.33 50.66 57.66 ~W 60 So 60 60 60 60 26*66 37*22 k2*22 65.55 75-55 93.88 99.43 11*10 27?77 36?10 57.77 67.22 84.43 96.10 * significant (P t = .05) bacteria applied after removal of reagent reagent and bacteria applied simultaneously » mean number cells infected • v , r n 7—r total number cells counted . \ (3 observations) io peritoneal macrophages at a concentration of 2.0 mM. 13 ' 97 ' 115 Experiments were conducted to test the effect of this compound upon the susceptibility of HIE cells to mycobacterial infection. Results indicated that dibutyryl cAMP significantly inhibited the infection of host cell monolayers at different concentrations dependent upon the infection procedure used (Table 2). It was found that 1.0 mM solutions caused significant inhibition with Procedure A. However, under Proce dure B, significant inhibition did not occur at concentrations less than 4.0 mM. Dibutyryl cAMP is soluble in the lipids of the host cell membrane, thereby allowing it to cross the intact cell membrane and inhibit phagocytosis. 31 It may be readily absorbed by M. avium due to the high-lipid content of its cell membrane, thereby impairing the ability of this compound to affect the HIE cells at lower concentra tions. Because phagocytosis is an energy-dependent process, experi ments were done to determine if disruption of carbohydrate metabolism affected the interaction between M. avium serotype 2 and HIE cells. Iodoacetate inhibits phagocytosis in PMN,^ blood monocytes,^ and peritoneal macrophages 74 ; in these cells, the energy required for phagocytosis has been linked to glycolysis which is known to be inhib ited by iodoacetate. 2,4-dinitrophenol inhibits Krebs' cycle activity and, thus phagocytosis, by alveolar macrophages. 74 These 2 compounds were tested for effects on mycobacterial infection of^H-IE cells. Tables 3 and 4 show results when iodoacetate and 2,4-dinitrophenol were employed, respectively. In both cases it was found that infection was Table 2. Effect of Dibutyryl cAMP on Interaction of M. avium Serotype 2 with HIE Cells. PROCEDURE At Bt Concentration mM X "* % 10.0 k.O 2.0 1.0 10 So 14.66 60 1*4.33 60 21.33 60 16V67 2V. ; i»3 23*88 35*56 0 10.0 4.0 2.0 1.0 57.66 60 11.66 60 26.66 60 33-33 60 46.33 60 59.33 60 96.10 19 V* 3 kb'lk 3 55.55 77.22 98.88 - significant (P = .05). t bacteria applied after removal of reagent T reagent and bacteria applied simultaneously ** mean number cells infected total number cells counted (3 observations) 0 Table 3. Effect of Iodoacetate on Interaction of M. avium Serotype 2 with HIE Cells. PRODECURE Af Concentration n*t X «'» 0.3 0.2 15.33 60 19.66 60 25*55 yi.ll 0.1 0.05 0.03 0.01 0 0.03 0.2 0.1 0.05 0.03 0.01 0 26 35.66 60 55 8.66 So 57.66 60 59.33 So So 18 27 SO 36.66 60 <14 So So 52.66 60 So 43*33 53M 91.67 96.10 98.83 1O13 30?00 45*00 16.10 73-33 87.77 98.33 59 ^significant (P » .05) t bacteria applied after removal of reagent ^ reagent and bacteria applied simultaneously mean number cells infected total number cells counted (3 observations) cn ro Table 4. Effect of 2,4-Dinitrophenol on Interaction of M. avium Serotype 2 with HIE Cells. PROCEDURE A+ Concentration 2.0 1.0 0.1 0.01 11.66 So" 27.66 60 29.33 60 35 19*43 46*10 48788 0.001 0 2.0 1.0 0.1 0.01 0.001 25 ^ 29-66 60 32.33 Sir 45.66 So 59-33 60 49743 53.88 76.10 98.88 mM 58.33 43-66 60 59 Zo 20.66 So" 72.77 98.33 34743 41767 * significant (P = .05). t bacteria applied after removal of reagent :[: reagent and bacteria applied simultaneously ** mean number cells infected total number cells counted (3 observations) cn CO 54 significantly reduced at a concentration of 0.10 mM or greater. Furthermore, there was no difference based upon the infection proce dure used. These data support other studies using cytochalasin B and dibutyryl cAMP by showing that the uptake of M. avium into intestinal epithelial cells is at least partially dependent upon the endocytic activity of the host cell. Bacterial Effects On Uptake Uptake of M. aviurn into HIE cells may be partially explained by factors related to the bacterium, such as its cell surface character istics (proteins or lipids) or metabolic products. This was studied using cultures of M. avium serotype 2 manipulated in various ways to alter these characteristics, and their subsequent interactions with HIE cells. Initial studies used bacteria killed by autoclaving or by suspension in formalin. Killed suspensions were incubated with HIE cells for 3 hours and the interaction was assessed quantitatively using the indirect fluorescent antibody stain. Figure 7 shows the results of incubation of autoclaved M. avium serotype 2 with HIE cell monolayers versus results from monolayers inoculated with viable M. avium cultures. A remarkable decrease in uptake was evident compared to the control, but a small amount of endocytosis (3.08%) did occur. Comparison of the results of methanol fixed and unfixed preparations (Figure 8), indicates that minimal engulfment probably reflects extracellularly adhered bacilli. Figure 9 shows the results of interactions of HIE cells with 55 100.0 90.0 8Q0 O vP a* $ 7 00 ® 60.0 *0 'C g 50.0 ca = 40.0 a> a u c 30.0 8 £ 20.0 10.0 AUTOCLAVED Figure 7. VIABLE Difference in Uptake of Autoclaved and Viable M. avium Serotype 2 by HIE Cells. Number of Associated Bacteria 51-60 UNFIXED FIXED 41-50 31-40 11-20 I-IO 100.0 80.0 60.0 40.0 20.0 0 20.0 40.0 60.0 BOO Percent HIE Cells/Monolayer Figure 8. Staining Characteristics of Methanol-Fixed and Unfixed HIE Cells Infected with Autoclaved M. avium Serotype 2. 100.0 100.0 90.0 ^ 800 |70.0 •M « 60.0 0> o 500 m = 40.0 d) o o 30.0 8 10.0 FORMALIN TREATED Figure 9. VIABLE Difference in Uptake of Formal in-Killed and Viable M. avium Serotype 2 by HIE Cells. formal in-killed and with viable M. avium. A significant inhibition (P = 0.05) in uptake of killed bacteria was found when compared to the control. Examination of methanol-fixed and unfixed preparations (Figure 10) indicated that the minimal level of endocytosis (3.79%) was more likely due to extracellular adhered bacteria than intra cellular bacteria, since the 2 preparations stain alike. Suspensions of M. avium serotype 2 were exposed to ultraviolet irradiation for 0 to 60 seconds and their interaction with HIE cells was compared to that of nonirradiated bacteria. An exposure of 40 seconds or more significantly reduced (P = 0.05) the infectivity of the organism (Figure 11). Results of methanol-fixed and unfixed prep arations were examined separately (Figure 12). After 40 seconds of irradiation preparations stained similarly indicating that the bacilli were not being taken into the host cell but were only adhered to the outside. After 50 to 60 seconds exposure the staining characteristics of methanol-fixed and unfixed preparations were identical. Viability tests on the M. avium suspensions paralleled those shown for infectivity in Figure 11. After 8 weeks of incubation at 37°C Middlebrook 7H10 Agar plates inoculated with suspensions exposed for 10, 20, or 30 seconds showed similar growth to plates inoculated with nonirradiated M. avium. Plates inoculated with bacterial cultures exposed for 40 seconds demonstrated a 65% reduction in growth compared to cultures containing nonirradiated bacilli. Plates inoculated with cultures exposed to 50 or 60 seconds of UV light exhibited no growth. The effects of short-term irradiation (less than 60 seconds) on mycobacteria are thought to be confined to the nucleic acids. 30 Number of Associated Bacteria 51-60 UNFIXED FIXED 41-50 31-40 21-30 11-20 1- 0 ) 1 1 I 100.0 8Q0 1- 60.0 I —1— 40.0 20.0 0 1 1 20.0 40.0 ... 1 1 1 60.0 80.0 loao Percent HIE Cells/Monolayer Figure 10. Staining Characteristics of Methanol-Fixed and Unfixed HIE Cells Infected with Formalin-Killed M. avium Serotype 2. 60 100.0 90.0 8Q0 S?70.0 •M ® 60.0 \ O V Q> O 50.0 m = 40.0 (V CJ o 30.0 I £ 20.0 10.0 10 20 30 40 50 Time (seconds) Figure 11. Difference in Uptake of M. avium Serotype 2 Irradiated with Ultraviolet Light (260 nm) for 0 to 60 Seconds. 60 1 NumberofAooctafed 1 20 sec. Number of Associated Bocterio 10 sec. Bocterio •«0 UNFIXED FIXED 1 |, 1 SMO 21-30 JJO | r ) -i. CO L—Ji i i * 40 20 0 i • 20 t i » •0(040200 PNcent HIE Cdls/Mooohiyer 20 40 CO «> Percent HIE Cells/Monolayer 1 80 10 I 40 sec. Number of Associated Bacteria 50 sec. Number of Associated Bocterio 91*60 UNFIXED 40 20 0 20 40 Percent HIE Grih/Monolayer UNFIXED 41-50 I 3M0 1 60 sec. Number of Associated Bocterio I1 51*60 4hS0 1 1 3MO FIXED » «-p L 1 • to • 40 20 * 0 • 20 40 Percent HIE Cells/Monoloyer Figure 12. i BO .i CO .i 40 .i 2D II 0 20 «0 Percent HIE Cells/Monolayer 60 90 . i SO 1 I. 60 1 i i 40 .i 20 0 i. —i 20 40 Percent HIE Cells/Monolayer Staining Characteristics of Methanol-Fixed and Unfixed HIE Cells Infected with M. avium Serotype 2 Irradiated with Ultraviolet Light for 0 to 60 Seconds. i . GO i 80 Therefore, it is likely that surface molecules would be unaltered in the suspensions irradiated in the present study. It may be inferred, from data obtained in these studies, that viability of the bacterium is an obligate requirement for endocytosis by HIE cells. The role of surface molecules in this process must be evaluated further. Although immunity to mycobacterial infections is cell-mediated rather than antibody-mediated, the effect of local antibody on the infection process in swine tuberculosis is unknown. Porcine anti-M. avium IgA is not available and, therefore, anti-M. avium serotype 2 serum (rabbit origin) was employed to evaluate the interaction of this organism with HIE cells. Results are shown in Figure 13. Inhibition of infection/attachment was inversely proportional to the concentration of antiserum. This may have resulted from the masking of surface structures which are required for attachment. Cultures of the anti- sera treated suspensions revealed no reduction of growth, implying that viability is not the sole requirement for M. avium uptake. Preliminary studies were conducted on the effect of milk and colostrum on uptake of M. avium by HIE cells. This was done to simu late conditions occurring in the gut of the neonate pig. Due to the extreme difficulty in obtaining milk and colostrum from sows, bovine milk and colostrum were substituted. Figure 14 indicates an increase in the number of internalized tubercle bacilli when M. avium was suspended in milk as compared to M. avium suspended in MEM. However, this increase was not statistically significant (P = 0.05). Figure 15 shows the results when epithelial cells were infected with M. avium 5Q0t— „— 400 O 15.0 I:256 164 l : 16 Concentration Figure 13. Interaction of M. avium Serotype 2 Suspended in Various Concentrations of Homologous Antiserum with HIE Cells. 64 100.0 90.0 ^ 8Q0 SB™ •« 60.0 \ o 'C Q> g 50.0 m = 40.0 a> u o V. "c 30.0 8 £ 20.0 10.0 MILK Figure 14. NO MILK Interaction of M. avium Serotype 2 Suspended in Bovine Milk or in MEM with HIE Cells. 65 100.0 90.0 8Q0 |70.0 •M ® 60.0 0> 8 50.0 m w = 40.0 0) o o 30.0 C 8V. <D CL 20.0 10.0 COLOSTRUM Figure 15. NO COLOSTRUM Interaction of M. avium Serotype 2 Suspended in Bovine Colostrum or in MEM with HIE Cells. 66 suspended in colostrum. A statistically significant (P = 0.05) increase in the percent of bacterial uptake was found when bacteria were suspended in colostrum. The difference in bacterial uptake between the 2 suspensions was possible due to their differences in lipid or immunoglobulin co'ntent. 22 - 54- 70 If pigs are exposed to M. avium during the neonatal period, not only are they physiologically incapable of preventing bacterial invasion of the gut, but their diet at this time may also enhance susceptibility to infection. This may indicate that sanitation during the first few days after birth could be of some importance in pre venting mycobacterial infection. CHAPTER 4 CONCLUSIONS An in vitro model using cultured Henle intestinal epithelial (HIE) cells was designed to study the early interaction between M. avium and the host cell in an attempt to better understand the patho- . genie mechanism involved in swine tuberculosis. The results of this study indicate that: 1. Maximum infection of cultured HIE cells was achieved after 3 hours of incubation with M. avium serotype 2. 2. Differences in characteristics among different serotypes of M. avium and saprophytic or nonpathogenic mycobacteria may account for the observed differences in infection rate with these organisms. 3. Because the rate of infection of HIE cells was inhibited by reagents shown to inhibit particle uptake in known phago cytic cells, it was suggested that uptake of M. avium by cultured epithelial cells was endocytic in nature. 4. Studies involving infection of HIE cells with dead M. avium showed that viability of the tubercle bacilli is necessary for endocytosis to occur. 5. Surface molecules may play a role in attachment of the organism to the host cell, but metabolic activity on the part of the tubercle bacilli is also required for endocy tosis. LIST OF REFERENCES 1. Allen, W. D., and Porter, P.: The Relative Frequencies and Distribution of Immunoglobulin-bearing Cells in the Intestinal Mucosa of Neonatal and Weaned Pigs and their Significance in the Development of Secretory Immunity. Immunol, 32, (1977): 819-827. 2. Allison, A. C., Davis, P., and DePetris, S.: Role of Contractile Microfilaments in Macrophage Movement and Endocytosis. Nature (London) New Biol, 232, (1971):153-155. 3. Angus, R. D.: Tuberculins For Use in Animal. Head, Bacterial Reagents, Veterinary Services Laboratories, APHIS (1977) USDA, Ames, IA 50010. 4. Axline, S. G., and Reven, E. P.: Inhibition of Phagocytosis and Plasma Membrane Mobility of Cultivated Macrophage by Cytochalasin B. J Cell Biol, 62, (1974):647-659. 5. Berthelson, J. D.: Economics of the Avian TB Problem in Swine. J Am Vet Med Assoc, 164, (1974):307-308. 6. Bickford, A. A., Ellis, G. H., and Moses, H. E.: Epizootiology of Tuberculosis in Starlings. J Am Vet Med Assoc, 149, (1966):312—318. 7. Bloch, H.: Studies on the Virulence of Tubercle Bacilli. Med, 91, (1950):197-218. 8. Bockman, D. E., and Cooper, M. D.: Pinocytosis by Epithelium Associated with Lymphoid Follicles in the Bursa of Fabricus, Appendix, and Peyer's Patches. An Electron Microscopic Study. Am J Anat, 136, (1978):455-478. 9. Buchanan, R. E., and Gibbons, N. E. Berguy's Manual of Deter minative Bacteriology. 8th ed. Baltimore, The Williams & Wilkins Company, (1974). J Exp 10. Chu, R. M., Glock, R. D., Ross, R. F., and Cox, D. F.: Lymphoid Tissues of the Small Intestine of Swine from Birth to One Month of Age. Am J Vet Res, 40, (1979):1713-1721. 11. Cline, M. J., and Lehrer, R. I.: Blood, 32, (1968):423-435. 68 Phagocytosis by Human Monocytes. 69 12. Cooper, M. S., Tuchberg, J. A., and Lifshitz, F.: Alterations in Rat Jejunal Permeability to a Macromolecular Tracer During a Hyperosmotic Load. Lab Inves, 38, (1978):447-453. 13. Cox, J. P., and Karnovsky, M. L.: The Depression of Phago cytosis by Exogenous Cyclic Nucleotides, Prostaglandins, and Theophylline. J Cell Biol, 59, (1973):480-490. 14. Crawford, A. B.: Studies in Avian Tuberculosis. Tuber, 37, (1938):594-597. 15. Dannenberg, A. M., Ando, M., Rojas-Espinosa, 0 . , Shima, K., and Tsuda, T.: Macrophage Activation in Tuberculous Lesions. Excerpta Med Intern, Congress, Series No. 325, (1973):223-235. 16. Davies, P., Fix, R. I., and Polyzonis, M.: The Inhibition of Phagocytosis and Facilitation of Exocytosis in Rabbit Poly morphonuclear Leukocytes by Cytochalasin B. Lab Invest, 28, (1973):16-22. 17. Derijcke, J . , Dervriese, L . , and Hoorens, J . : An Outbreak o f Avian Tuberculosis in Pigs. Vlaams Dieraenleskundig Ti jdschrift, 45, (1976):1-8. 18. Feldman, W. H.: Avian Tuberculosis Infections. Wi1 kins Company, (1938): Baltimore, MD. 19. Feldman, W. H.: Generalized Tuberculosis of Swine Due to Avian Tubercle Bacilli. J Am Vet Med Asso, 92, (1938):681-684. 20. Feldman, W. H.: Tuberculosis. In: Diseases Transmitted from Animals to Man. 5th ed. Edited by T. G. Hull. Springfield, IL. Charles C. Thomas, (1963):5-81. 21. Feldman, W. H., Moses, H. E., and Karlson, A. G.: Corynebacterium equi as a Possible Cause of Tuberculous-like Lesions of Swine. Cornell Vet, 30, (1940):456-461. 22. Fleenor, W. A., and Stott, G. H.: Single Radial Immunodiffusion Analysis for Quantitation of Colostral Immunoglobulin Concen tration. J Dairy Sci, 64, (1981):740-747. 23. Fodstad, F. H.: Tuberculin Reactions in Bulls and Boars Sensitized With Atypical Mycobacteria from Sawdust. Acta Vet Scand, 18, (1977):374-383. 24. Fukuyama, K., Junkichi, T., and Masahiko, K.: Effect of Cord Factor (Mycobacterial Toxic Glycolipid) on Liver Microsomes in vivo. J Biochem, 69, (1971):511-516. Amer Rev The Williams & 70 25. Gilmour, N. J. L.: The Specificity of the Fluorescent Antibody Test in Rabbits Inoculated with M. avium, M. johnei, and M. bovis var. BCG. Res Vet Sci, 12, (1971):295-297. 26. Goren, M. B.: Phagocyte Lysosomes: Interactions with Infectious Agents, Phagosomes, and Experimental Pertubations in Function. Am Rev Microbiol, 31, (1977):507-533. 27. Goren, M. B., Brokl, 0., and Schaefer, W. B.: Lipids of Putative Relevance to Virulence in Mycobacterium tuberculosis: Correlation of Virulence with Elaboration of Sulfatides and Strongyl Acidic Lipids. Infect Immun, 9, (1974):142-149. 28. Goslee, S., and Wolinsky, E.: Water as a Source of Potentially Pathogenic Mycobacteria. Am Rev Resp Dis, 113, (1976):287290. 29. Graham, R., and Tunnicliff, E. A.: Fowl Tuberculosis in Swine. Trans Illinois Acad Sci, 19, (1926):138-143. 30. Hale, T. L., and Bonventre, P. F.: Shigella Infection of Henle Intestinal Epithelial Cells: Role of the Bacterium. Infect Immun, 24, (1979):879-886. 31. Hale, T. L., Morris, R. E., and Bonventre, P. F.: Shigella Infection of Henle Intestinal Epithelial Cells: Role of the Host Cell. Infect Immun, 24, (1979):887-894. 32. Hartlova, E., and Mohelska, H.: Reliable Technique of Prepara tion of Ultrathin Section of Tissue Infected with Mycobacteria. Folia Micro, 23, (1978):406-411. 33. Hartwig, J. H., and Stossel, T. P.: Interaction of Actin, Myosin, and a New Actin Binding Protein of Rabbit Pulmonary Macrophages. I I I . Effects of Cytochalasin B. J Cell Biol, 71, (1976):295-303. 34. Hignett, S. L., and McKenzie, D. A.: The Occurrence of Tubercu losis in the Starling. Vet Rec, 52, (1940):585-588. 35. Horn, L. H.: Cost of TB on the Swine Industry. Symposium on Swine Mycobacterial Infections. Madison, WI (September 30 October 1, 1975). St. Paul, MN: Livestock Conservation Institute. 36. Huitema, H., and Jaartsveld, F. H. J.: M. microti Infection in a Cat and Some Pigs. Antonie Van Leuvenhook. J. Microbiol Serol, 33, (1968):209-212. 71 37. Jarnigan, J. L., Richards, W. D., Muhm, R. L., and Ellis, E. M.: The Isolation of M. xenopi from Granulomatous Lesions in Swine. Am Rev Resp Dis, 104, (1971):763-765. 38. Jones, R. J., and Jenkins, D. E.: Mycobacteria Isolated From Soil. Can Jour Micro, 11, (1965):127-130. 39. Jones, W. D., Saito, H., and Kubica, G. P.: Fluorescent Antibody Techniques with Mycobacteria. Am Rev Resp Dis, 92, (1965):255260. 40. Karlson, A. G.: The Genus Mycobacterium. In: Veterinary Bacteriology and Virology. 7th ed. Edited by I. A. Merchant, and R. A. Packer. Iowa State University Press, Ames, IA (1967). 41. Karlson, A. G.: Tuberculosis - Isolation and Identification of Avian Pathogens. Amer. Assoc. Avian Pathologists. Dept. Vet. Micro., Texas A & M University, College Station, Texas (1975): 99-108. 42. Karlson, A. G., Davis, C. L., and Cohn, M. L.: Skotochromogenic Mycobacterium avium from a Trumpeter Swan. Am Jour Vet Res, 23, (1962):575-578. 43. Karlson, A. G., Moses, H. E., and Feldman, W. H.: Corynebacterium equi in the Submaxillary Lymph Nodes of Swine. Infect Dis, 67, (1941):243-251. J 44. Krasnow, I., and Wayne, L. G.: Comparison of Methods for Tuberculosis Bacteriology. App Micro, 18, (1969):915-923. 45. Kato, M., and Fukushi, K.: Studies of a Biochemical Lesion in Experimental Tuberculosis in Mice. X. Mitochondrial Swelling Induced by Cord Factor in vivo and Accompanying Biochemical Change. Am Rev Respir Dis, 100, (1969):42-46. 46. Kato, M., and Goren, M. B.: Synergistic Action of Cord Factor and Mycobacterial Sulfatides on Mitochondria. Infect Immun, 10, (1974):733-741. 47. Kato, M., Kusunose, M., Miki, K., Matsunaga, K., and Yamamura, Y . : The Mechanism o f the Toxicity o f Cord Factor. Amer Rev Tuberc, 80, (1959):240-248. 48. Kelley, E. M.: Department of Microbiology, University of Arizona, Tucson. 49. Kihlstrom, E.: Infection of HeLa Cells with Salmonella typhimurium 395 MS and MR10 Bacteria. Infect Immun, 17, (1977):290-295. 72 50. Kingham, J. G. C., Whorwell, P. J., and Loehry, C. A.: Small Intestinal Permeability: Effects of Ischaemia and Exposure to Acetyl Salicylate. Gut, 17, (1976):354-361. 51. Kubicn, M., and Matuskova, E.: Serological Typing of Myco bacteria for Tracing Possible Sources of Avian Mycobacterial Infections in Man. Bull Wld Hlth Org, 39, (1968):657-659. 52. Kusunose, E., Ichihara, K., Noda, Y., and Kusunose, M.: Superoxide Dismutase from Mycobacterium tuberculosis. Biochem, 80, (1976):1343-1352. J 53. Lecce, J. G.: Glucose Milliequivalents Eaten by the Neonatal Pig and Cessation of Intestinal Absorption of Large Molecules (Closure). J Nutrition, 90, (1966):240-253. 54. Lecce, J. G.: Effect of Dietary Regimen on Cessation of Uptake of Macromolecules by Piglet Intestinal Epithelium (Closure) and Transport t o the Blood. Proc Soc Amer Microbiol, 72, (1972):234-241. 55. Lederer, E.: Cord Factor and Related Trehalose Esters. Phys Lipids, 16, (1976):91-106. 56. Lesslie, I. W., and Birn, D.: Correlation of Biological Potency of Human and Bovine Tuberculin PPDS in Guinea Pigs, Cattle, and Man. 0 Biol Stan, 4, (1976):39-42. 57. Livestock and Veterinary Sciences. Annual Report of the National Research Programs (1978). 58. Luke, D.: Studies in Tuberculous Sensitivity in the Pig. I I I . The Tuberculin Skin Reaction i n the Sow. Vet Rec, 64, (1952):344-345. 59. Mailman, W. L.: Should Tuberculosis be Eradicated from all Species? Health Lab Sci, 9, (1972):133-139. 60. Mailman, W. L., and Mailman, V. H.: Natural and Experimental Infectivity of M. avium Complex Microorganisms in Swine. Symposium on Swine Mycobacterial Infection. Madison, WI (September 30 - October 1, 1975). St. Paul, MN: Livestock Conservation Institute. 61. Mailman, W. L., and Mailman, V. H.: Natural and Experimental M. avium Complex Microorganisms in Swine. Symposium on Swine Mycobacterial Infections. Madison, WI (September 30 October 1, 1975). St. Paul, MN: Livestock Conservation Institute. Chem 73 62 Mailman, V. H., and Mailman, W. L.: Relationship of Atypical Bovine and Porcine Mycobacteria to Those of Human Origin. Health Lab Sci, 1, (1964):11-15. 63 McCarter, J., Beach, B. A., and Hastings, E. G.: The Relation of the Avian Tubercle Bacillus to TB in Swine and Incidentally in Cattle. J Am Vet Med Assoc, 86, (1935):168-171. 64 McCarthy, C.: Effect of Palmitic Acid Utilization on Cell Division i n Mycobacterium avium. Infect Immun, 9 , (1974): 363-372. 65 McFarland, W., and Hellman, D. H.: Comparison of Lymphocytes Transformation and Intradermal Reactions to Tuberculins. Am Rev Resp Dis, 93, (1966):742-747. 66 McGavin, M. D., Mailman, V. H., and Mailman, W. L.: Lesions and Tuberculin Sensitivity i n Calves Inoculated with Group I I I Mycobacterial Isolates from Swine, Pen, Soil, and Cattle Feed. Am J Vet Res, 36, (1975):641-646. 67 Meat and Poultry Inspection Regulations. Meat and Poultry Inspection Program. Food Safety and Quality Service, USDA. Washington, D.C. (1982):44-45. 68 Meissner, G., and Anz, W.: Sources of Mycobacterium avium Complex Infection Resulting in Human Diseases. Am Rev Resp Dis, 116, (1977):1057-1059. 69 Mitchell, M. D., Huff, I. H., Thoen, C. 0., Himes, E. M., and Howder, J. D.: Swine Tuberculosis in South Dakota. J Am Vet Med Assoc, 167, (1975):152-156. 70 Murata, H., and Namioka, S.: The Duration of Colostra! Immuno globulin Uptake by the Epithelium of the Small Intestine of Neonatal Piglets. J Comp Path, 87, (1977):431-438. 71 Muscoplat, C. C., Thoen, C. 0., Chen, A. W., Rakick, P. M., and Johnson, D. W.: Development of Specific Lymphocyte Immunostimulation and Tuberculin Skin Reactivity in Swine Infected With M. bovis and M. avium. Am J Vet Res, 36, (1975):1167-1171. 72 Nasseri, D. L., Kiumarss, J. L., and Eveland, W. C.: Specific Detection of Mycobacteria by the Immunofluorescent Technique. Int J Zoon, 4, (1977):31-37. 73 Noll, H., Bloch, H., Asselineau, J., and Lederer, E.: The Chemical Structure of the Cord Factor of Mycobacterium tubercu losis. Biochem Biophys Acta, 20, (1956):299-309. 74 74. Oren, R., Farnham, A. E., Saito, K., Milofsky, E., and Karnovsky, M. L . : Metabolic Patterns i n Three Types of Phagocytizing Cells. J Cell Biol, 17, (1963):487-501. 75. Owen, R. L.: Sequential Uptake of HRP by Lymphoid Follicle Epithelium of Peyer's Patches in the Normal Unobstructed Mouse Intestine. An Ultrastructural Study. Gastroenterology, 72, (1977):440-451. 76. Owen, R. L., Allen, C. L., and Stevens, D. P.: Phagocytosis of Giardia muris by Macrophages in Peyer's Patch Epithelium in Mice. Infect Immun, 33, (1981):591-601. 77. Owen, R. L., and Jones, A. L.: Epithelial Cell Specialization Within Human Peyer's Patches: An Ultrastructural Study of Intestinal Lymphoid Follicles. Gastroenterology, 66, (1974): 189-203. 78. Owen, R. L., and Jones, A. L.: Specialized Lymphoid Follicle Epithelial Cells in the Human and Nonhuman Primate: A Possible Antigen Uptake Site. SEM, Part III. Proceedings of the Workshop on Advances in Biomedical Applications of the SEM I IT Research Institute. Chicago, IL (1974). 79. Owen, R. L., and Nemanic, P.: Antigen Processing Structures of the Mammalian Intestinal Tract: An SEM Study of Lymphoepithelial Organs. Scanning Electron Microscopy, 11, (1978): 367-372. 80. Perryman, B. S., Spitznagel, J. K., and Becker, C.: Observations in a Swine Herd Infected with Mycobacterium Complex Species and Possible Interaction of the Herd with the Environment. Symposium on Swine Mycobacterial Infections. Madison, WI. (September 30 - October 1, 1975) St. Paul, MN: Livestock Conservation Institute. 81. Pritchard, W. D.: Outbreak of Mycobacterium avium Infection in a Swine Herd in Idaho. Symposium on Swine Mycobacterial Infections. Madison, WI. (September 30 - October 1, 1979) St. Paul, MN: Livestock Conservation Institute. 82. Raffel, S.: The Mechanism Involved in Acquired Immunity to Tuberculosis. In: Experimental Tuberculosis. Edited by G. E. W. Wolstenholme, M. P. Cameron, and C. M. O'Connor. Boston, Little, Brown and Company. (1955):131-159. 83. Ray, J. A., Mailman, V. H., Mailman, W. L., and Morrill, C. C.: Pathologic and Bacteriologic Features and Hypersensitivity of Piqs Given M. bovis, M. avium, or Group III Mycobacteria. Am J Vet Res, 33,TT972): 1333-1345. 75 84. Reznikov, M., and Leggo, J. H.: Examination of Soil in the Brisbane Area for Organisms of the Mycobacterium AviumIntracellulare-Scrofulaceum (MAIS) Complex. Pathology, 6, (1974):269-273. 85. Reznikov, M., and Robinson, J.: Serologically Identical Battey Mycobacteria From Sputa of Healthy Piggery Workers and Lesions of Pigs. Aust Vet J, 46, (1970):606-609. 86. Runyon, E. H.: Mycobacterium intracellular. 95, (1967):861-865. 87. Sakamoto, A.: Anatomy and Physiology of the Small Intestine of Swine. Independent Study. University of Arizona, Tucson. (1978). 88. Savage, D. C.: Survival of Mucosal Epithelia. Epithelial Pene tration and Growth in Tissues of Pathogenic Bacteria. Symp Soc Gen Microbiol, 22, (1972):25-57. 89. Savage, D. C.: Electron Microscopy of Bacteria Adherent to Epithelia in the Murine Intestinal Canal. In: Microbiology 1977. Edited by D. Schlessinger. American Society for Microbiology. (1977):422-443. 90. Sbarra, A. J., and Karnovsky, M. L.: The Biochemical Basis of Phagocytosis. I. Metabolic Changes During the Ingestion of Particles by Polymorphonuclear Leukocytes. J Cell Biol, 234, (1959):1355-1362. 91. Schaefer, W. B.: Incidence of the Serotypes of Mycobacterium avium and Atypical Mycobacteria in Human and Animal Diseases. Am Rev Resp Dis, 97, (1967):18-23. 92. Shramek, G., Falk, L., Jr.: Diagnosis of Virus-Infected Cultures With Fluoresce!'n-Labeled Antiserum. In: Tissue Culture Methods and Applications. Edited by P. F. Kruse, Jr., and M. K. Patterson, Jr. Academic Press Inc., New York. (1973):532-535. 93. Snellen, J. E., and Savage, D. C.: Freeze-Fracture Study of the Filamentous Segmented Microorganism Attached to the Murine Small Bowel. J Bacterid, 134, (1978):1099-1109. 94. Sodeman, W. A.: Sodeman's Pathologic Physiology. In: Mechanisms of Disease. 6th ed. Edited by W. A. Sodeman, Jr., and T. M. Sodeman. Philadelphia: Saunders, (1979). 95. Songer, J. G., and Bicknell, E. J.: Involvement of Wood Shavings Used as Bedding in the Epidemiology of Swine Tuberculosis. 59th Conference of Research Workers in Animal Disease, Abstract No. 70. Am Rev Resp Dis, 76 96. Songer, J. G., Bicknell, E. J., and Thoen, C. 0.: Investigation of Swine Tuberculosis in Arizona. Med, 44, (1980):115—120. Epidemiology Can J Comp 97. Stossel, T. P., Mason, R. J., Hartwig, J., and Vaughan, M.: Quantitation Studies of Phagocytosis by Polymorphonuclear Leukocytes: Use of Emulsions to Measure the Initial Rate of Phagocytosis. J Clin Invest, 51, (1972):615-624. 98. Suter, E.: Passive Transfer of Acquired Resistance to Infection with Mycobacterium tuberculosis by Means of Cells. Am Rev Resp Dis, 83, (1961):535-542. 99. Suter, E., and Ramseier, H.: Cellular Reactions in Infection. Adv Immunol, 4, (1964):117-128. 100. Tammemagi, L., and Simmons, G. C.: Battey-Type Mycobacterial Infection of Pigs. Aust Vet Jour, 44, (1967):121-124. 101. Thoen, C. 0., Armbrust, A. L., and Hopkins, M. P.: Enzyme-linked Immunosorbent Assay for Detecting Antibodies in Swine Infected with Mycobacterium avium. Am J Vet Res, 40, (1979):1096-1099. 102. Thoen, C. 0., and Himes, E. M.: Isolation of M. chelonei from a Granulomatous Lesion in a Pig. Jour Clin Micro, 6, (1977): 81-83. 103. Thoen, C. 0., Himes, E. M., and Barrett, R. E.: Mycobacterium avium Serotype 2 Infection in a Sandhill Crane. Jour Wildlife Dis, 13, (1977):40-43. 104. Thoen, C. 0., Himes, E. M., Weaver, D. E., and Spangler, G. W.: Tuberculosis in Brood Sows and Pigs Slaughtered in Iowa. Am J Vet Res, 37, (1976):775-781. 105. Thoen, C. 0., Jarnigan, J. L., and Richards, W. D.: Isolation and Identification of Mycobacteria from Porcine Tissues: A Three-Year Summary. Am J Vet Res, 36, (1975):1383-1386. 106. Thoen, C. 0., Johnson, D. W., Himes, E. M., Menke, S. B., and Muscoplat, C. C.: Experimentally Induced M. avium Sero 8 Infection in Swine. Am J Vet Res, 37, (1976):177-181. 107. Thoen, C. 0., and Karlson, A. G.: Tuberculosis. In: Diseases of Poultry. 7th ed. Edited by M. S. Hofstad, B. W. Calnek, C. F. Termboldt, W. H. Reid, and H. W. Yoder, Jr. Iowa State University Press, Ames, IA. (1978):209-225. 108. Thoen, C. 0., and Karlson, A. G.: Swine Tuberculosis. In: Diseases of Swine. 5th ed. Edited by A. D. Leman, R. D. Glock, 77 W. L. Mengeling, R. H. C. Penny, E. Scholl, and B. Straw. State University Press, Ames, IA. (1981):712-729. Iowa 109. Thoen, C. 0., Karlson, Swine Tuberculosis. tions. Madison, WI. Paul, MN: Livestock A. G., and Himes, E. M.: Diagnosis of Symposium on Swine Mycobacterial Infec (September 30 - October 1, 1975) St. Conservation Institute. 110. Thoen, C. 0., Richards, W. D., and Jarnigan, J. L.: Mycobacteria Isolated from Exotic Animals. J Am Vet Med Assoc, 190, (1976): 987-990. 111. Thomas, D. W., Hill, J. C., and Tyeryar, F. J.: Interaction of Gonococci with Phagocytic Leukocytes from Men and Mice. Infect Immun, 8, (1973):98-104. 112. Van Es, L.: Nebraska. 113. Walker, W. A.: Immunological Mechanisms Controlling Macromolecular Penetration in the Small Intestine. In: Microbiology - 1975. Edited by D. Schlessinger. American Society for Microbiology. (1975):158-163. 114. Walker, W. A., and Isselbacher, I.: Phys in Med, 297, (1977):767-789. 115. Welscher, H. D., and Crudehaud, A.: The Relationship Between Phagocytosis, Release of Lysosomal Enzymes and 3'5' Cyclic Adenosine Monophosphate in Mouse Macrophages. Adv Exp Med Biol, 66, (1975):705-710. 116. Wolinsky, E.: Mycobacteria. In: Microbiology. 3rd ed. Edited by B. D. Davis, R. Dulbecco, H. N. Eisen, H. S. Ginsberg. Harper and Row. (1980):724-740. 117. Wolinsky, E., and Schaefer, W. B.: Proposed Numbering Scheme for Mycobacterial Serotypes by Agglutination. Int J Syst Bact, 23, (1973):182-183. 118. Youmans, G. P.: Nature of the Specific Acquired Immune Response in Tuberculosis. In: Tuberculosis. W. B. Saunders Company, Philadelphia, PA. (1979):285-298. 119. Zigmond, S. H., and Hirsch, J. G.: Effects of Cytochalasin B on Polymorphonuclear Leukocyte Locomotion, Phagocytosis and Glycolysis. Exp Cell Res, 73, (1975):383-393. Tuberculosis of Swine. Lincoln University of (1925). Agric. Exp. Stn. Circ. 25. Intestinal Antibodies.