ABSTRACT ULTRASTRUCTURAL AND HISTOCHEMICAL STUDIES OF MYCOPLASMA INFECTED TURKEY SINUS EPITHELIAL CELLS by Grant Wayne Boam Fourdweek—old turkey poults were injected into the infraorbital sinuses with 0.2 ml. broth culture of Mycoplasma gallisepticum S-6 strain. Infected and control birds were killed"each week for 6 weeks. The epithelial lining cells of the infraorbital sinuses were stained to demonstrate the activity of selected enzymes from the hydrolytic, proteolytic, and oxidative groups. Only enzymes characteristic of basic energy—generating pathways were found-—succinic acid, isocitric acid, lactic acid, and malic acid dehydrogenase. Enzyme activity did not increase or decrease with infection, epithelial cell hyperplasia, or increase in host age from 5 to 10 weeks. The mchplasma—infected cells were also examined with an electron microscope. The organisms were found in large numbers during the first week of infection but were fewer in number by the sixth week. The infection was uncomplicated by secondary invaders during the first week but, by the sixth week, bacteria and fungal Spores had appeared. .uyco- plasmas were lying between cells and within cells, but few were free in the exudate. The nuclei were only minimally affected; they were apparently pressed toward the base of the cell. The mitochondria and ribosomal structures of infected cells disappeared from the cytoplasm. The cilia and microvilli were lost from the surface of heavily infected cells but not from adjacent uninfected cells. ULTRASTRUCTURAL AND HISTOCHEMICAL STUDIES OF MYCOPLASMA INFECTED TURKEY SINUS EPITHELIAL CELLS By Grant Wayne Boam A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pathology 1970 v-n' v I wish professor as during this thanks; to “m 0f the in "Y acacia 3r.c. C. M State Unive fafilities’ For su ACKNOWLEDGEMENTS I wish to express my appreciation to Dr. Vance L. Sanger, major professor and honored associate, for his guidance and encouragement during this project. Also to my academic committee I wish to express thanks: to Dr. Gordon Carter for his help and guidance in the culture work of the mycoplasma organism, to Dr. Allan Trapp for encouragement in my academic program and valuable advice on publications, and to Dr. C. C. Morrill, Chairman of the Department of Pathology, Michigan State University, for providing the electron microsc0pe, other research facilities, and funds that made this project possible. For supplemental financian support in this work, a special thanks and acknowledgement is extended to Dr. J. Hoefer, Associate Director, Agricultural Experiment Station, Michigan State University. A helping hand was always extended to me by Dr. Gordon Spink and Mrs. June P. Mack of the campus electron microscOpe laboratory, Michigan State University. I am most grateful for their help with the electron microsCOpy portion of this project. I wish to thank Dr. Ester Roege for the services she most graciously rendered in sectioning and preparing the grids for electron microscOpic axamination . A special kind of gratitude, that is not always adequately expressed, I Vish to extend to my wife, Sharon, and the children, Karine, Bryan and Jan. Their patience and encouragement make all academic endeavors worth While . ii INTRODUCTION. . LITERATURE REVIEW . MATERIALS AND METHODS RESULTS . . . . . . . DISCUSSION. . . . . . SUMMARY . . . . . . . REFERENCES. . . . . . APPENDICES. . . . . . VITA I O O O O O O 0 TABLE OF CONTENTS iii Page 18 25 SO 56 57 69 84 Table LIST OF TABLES Page Enzyme, reaction catalyzed, control tissues and references for eleven enzymes . . o . o . . . o c . o . . c . . 0 . o . 24 Enzyme activity of control and myc0plasma—infected epithelial cells in turkey infraorbital sinus . . . . . . . . c . a c 49 iv Figure 10 ll 12 l3 14 15 16 17 18 19 20 21 22 LIST OF FIGURES Front view of a turkey infected with Mycoplasma gaZZiSQPtiCu/n 8—60 0 o o o o o o e o e c o 0 Side view of a turkey infected with Myccplasma gallisepticum S-6. . . . . . . . . . . . . . . . Outside surface of sinus skin covering . Inside surface of sinus and mucous membrane lining . Infected turkey-—first week. x 6,800 . . Infected turkey-—first week. x 13,600. . . . . . . Infected turkey--first week. x 20,400. . . . . . . Infected turkey——first week. x 9,520 . . . . . . . Infected turkey-~first week. x 4,740 . . . Infected turkey-—first week. x 6,800 . . . . . . Infected turkey——first week. x 13,600. . . . . . Infected turkey--first week. x 6,800 . Control turkey--first week. x 6,800. . . . . . . . Infected turkey—-sixth week. x 9,520 . . . . . . . Infected turkey--first week. x 6,800 . . . . . . . Infected turkey--sixth week. x 20,400. . . . . . . Infected turkey—-sixth week. x 6,400 . . . U . . Infected turkey--first week. x 6,800 . Succinic acid dehydrogenase. x 550 . . . . . . . . Isocitric acid dehydrogenase. x 550, . . . . . . . Malic acid dehydrogenase . . . , . . . . . . . . . Lactic acid dehydrogenase. . . . . . . . . . . . . ‘ v 30 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 46 46 48 48 LIST OF APPENDICES Appendix A Myc0plasma culture media. . . . . . . . B Fixation. . . . . . . . . . . . . . . . Glutaraldehyde . . . . . . . . . Osmium tetroxide . . . . . . . C Embedding . . . . . . . . . . . . . . . D Staining. . . . . . . . . . . . . . . . Thick sections . . . . . . . . . Reynolds lead citrate stain. . . Uranyl acetate stain . . . . . . E Kodak electron image plate deveIOpment. vi Page 69 70 70 71 72. 78 78 79 80 81 INTRODUCTION The avian infraorbital sinuses are shallcw cavities lined by pseudo- stratified, ciliated, columnar epithelium with abundant goblet cells. The epithelial cells of the mucous membrane rest on a thin basement meme brane and lamina prOpria. Healthy sinuses are empty except for small amounts of mucus to moisren the surface. Because of their anatomical location, the main stream of air does not pass over these epithelial surfaces during respiration. Since the surface lining cells do not actively participate in gas exchange, it seems reasonable to assume that the cellular metabolic activity would be little more than ensugh to maintain the biolotical integrity of the mucous membrane. With Mycgplasma gallisepttcum infection in turkeys, however, extensive changes occur in the sinuses and lining membranes. The sinus fills with mucous exudate, the epithelial cells undergo hyper- trophy and hyperplasia, and mononuclear inflammatory cells accumulate in the submucosa. There is little evidence of necrosis in the early stages. The body of research work on this disease, its clinical appearance, histopathology, and treatment is extensive. The cultural, cellular and subcellular characteristics of the organism have also been widely studied. The objectives of this study were to: (1) describe the pathogenesis of mchplasma infection in the turkey sinus epithelial cell by use of the electron microsc0pe, and (2) ccrrelate the progress of the disease 2 with changes, if any, in the enzymatic composition of the cells. It is known that the stresses on cellular systems will cause induction of enzymes not normally produced by those cells, or toxins may cause destruction of the cell's capabilities to produce enzymes needed for metabolism. These studies were conducted using young susceptible turkey poults with experimentally produced infections. LITERATURE REVIEW Avian Mycoplasmosis- History. A disease in turkeys similar to that which is now called chronic respiratory disease (GED) or infectious turkey sinusitis,(ITS) was first described by M‘Fadyean (1893). Dodd (1905) made a more detailed description of:a similar disease in 7 turkeys from one.farm, with epizootic pneumoenteritis. The first accurate description of CRD in the United States was made by Tyzzer (1926). He_also suggested the use of argyrol (4% silver nitrate) injections into the sinus as a treatment . Delaplane at al. (1933) reported a_high incidence of a.rapidly spreading respiratory disease in turkeys of the northeastern.United States. It was not until\1938, however, that ITS was identified by Dickinson and Hinshaw_as a separate disease distinguishing it from vitamin A deficiency,and fowl corysa. There are conflicting reports in the literature that the entire- syndrome.of avian upper-respiratory disease was caused by a bacterium. . Eliot and.Lewis (1934) reporteda."henophilic".bacterium which is probably the same organism isolated by Page et a2. (1963) and considered to be, the causative agent of true fowl coryza. Nelson (1935) described coccobcailliform bodies associated with sinus infections of.turkeys. The next year he cultured a filterable organism in egg embryos and tissue culture (Nelson, 1936). This was 4 probably the same organism now known-to cause CRD in turkeys and chickens described by Van Rockeland Gray (1957). Chronic respiratory disease was named and described bleelaplane and Stuart in 1943. They suggested that the agent was viral—like in its characteristics. Groupé-et al. (1948, 1949) isolated the "turkey sinusitis organism" from chick embryos. Delaplane (1948, 1949) described the lesions pro— duced-in the chick embryos which be attributed to a virus. This work was verified and expanded by Chute (1953, 1954). He described hemorrhages. of the skin of the embryo and of the amnionic and yolk sac membranes. The embryo was stunted in size and had enlargements of the joints of; the legs, wings, and articulations of the mandible. In 1952, Van Roekel et a2. used-all the cultural procedures, except the tissue culture method of Nelson, to study CRD. A pure culture of organisms.from ITS was serially passaged in chicken eggs. Hhen.yolk cultures_were injected into chicken_sinuses a.condition similar to sinu- sitis in turkeys was produced. Markaham and Wong (1952) succeeded in isolating pleuropneumonia-like organisms (PPLO) from exudates of chickens and turkeys with CRD using serumrenriched cell-free medium. By 1960 it was well established that the organism causing CRD and ITS was the same (Van.Roekel and Olesiuk, 1953; Osborn and Pomeroy, 1958; Adler, 1960a). Freundt,(1960) classified the mchplasms on the basis of _morphology and species while Edward_and Kandrck (1960) named the causative organism of ITS, M. galliseptioum. The most common strain used in research is the 8-6 mutant of Zander (1961). Yoder and Hofstad (1964) reviewed the characterization oftmchplasma found in the avian species. MyoopZaQMa gallissptioum is often found in pure culture in the sinuses; however, complex infection with multiple microbial agents has 5 been reported (Biddle and Cover, 1957; Adler er al., 1962; Blake, 1962; Glantz, 1962). Culture. After the initial cell-free culture of M. gallisspricum by Markaham and Wong (1952), many reports of successful prOpagation of organisms on artificial media began to appear in the literature (Grumbles et al., 1953; Adler at al., 1954; Hofstad and Doerr, 1956; Taylor and Fabricant, 1957, 1958; Adler and Berg, 1960). The organisms cultivated on artificial media were used for serologic studies and early investiga— tions for vaccine production (Yamamoto and Adler, 1958; Hall, 1962). They were used for biochemical and metabolic studies, and also to study growth and reproduction (Ruys st cl., 1967; Razin at aZ., 1967b). Hayflick and Stinebring (1960) and Hayflick (1965) reported the growth and appearance of mycoplasma in tissue culture. The myc0plasma species infecting cell cultures have often led to miSinterpretation of data if their presence was unknown. Dmochowski at al. (1967) described cultural characteristics on cell— free media and pleomorphism of these organisms by electron microscopic examination. Serology. Jungherr at al. (1953) and Fahey at al. (1954) first used sero- logical studies to diagnose CRD. Gianforte et a1. (1955) and Moore et a2. (1960) are only 2 of the many research groups that have studied the development of the disease in infected birds with serum assays. The Salsbury Laboratories* have deveIOped a commercially available plate test antigen for mycoplasma screening. *Salsbury Laboratories, Charles City, Iowa. 6 Many workers both in the laboratory and in field trials (Fabricant and Levine, 1953; Olsen at aZ., 1964; Olsen at aZ., 1962; McMartin and Adler, 1961; Adler at al., 1960; Adler, 1960b; Domermuth, 1962; Domermuth, 1957; Olesiuk and Van Roekel, 1960) have demonstrated an immune response after infection with M1 gallisspticum. From this work it is evident that the strain of organisms used, the number of organisms, and the route of inoculation were all important factors in quantitating the response. Adler et a1. (1960) suggested the eXperimental infection of young poults to build a life-long immunity. Luginbuhl at al. (1967) studied the advantages and disadvantages of infecting young birds. He reported vac- cine efficacy, carrier state, passive antibody transfer to the young bird and persistence of antibody, and concluded that limited use of vac— cination was advisable. Transmission. EXperimentally the disease can be produced by eXposing young animals to the myc0plasma by unnatural routes, such as swabbing the trachea, intramuscular injection, and others (Jerstad at aZ., 1950). It is generally believed that natural transmissions of the organisms are airborne, which may be influenced by climatic conditions (Fahey and Crawley, 1955). Transmission of the mycoplasma through the egg is pos- eible but inconsistent (Jerstad at al., 1959a,b). Abbot (1960) and Kumar (1963) have made additional reports of egg transmission and have stressed the economic importance of this mode of.transmiasion in prevent- ing eradication. Several methods of killing the organism in the egg have been suggested (Mataney at aZ., 1955). Hoyt at al. (1952) used sulfamethazine, crystal violet and antibiotics. Horowitz and Maniloff (1967) described the life cycle of the organism in and out of the host animal. Osborn and Pomeroy (1958) list turkeys, 7 chickens, ducks, pheasants, guinee fowl, pigeons, partridges, peacocks, and cotton rats as host species offlM; gallisepticum with Swiss mice and Norway.rets refractory. Madden.et a1. (1967) isolatede, gaZZisepticum from e.Bob-Hhite quail. Electron microscopy. The electron microscopic reports on.M;.gaZZ£sep- tummy describe the organism and its structure (White at aZ.,‘1954; Horowitz at 4.1,, was; Dmochowski, a; «z. , 1967; Mandloffet a2. , 1965), its colonia1~grewth.(Shifrine et al.. 1962) and the structures of the organism as they relate to the function (Maniloff and'Morowite, 1967; Resin, 1967a). Reagan (1953) compared subcelluler characteristics of A strains-of,CRB agents.- A general electron microscopic survey of mycoplasma species was made by Domernuth at al. (1964). Edwards and Fogh (1960) studied the fine structure of.mycoplaeme in.tissue culture and Chu and Horne (1967) compared several mycoplasma species with similar appearing fit“; 38 9. Hietochggietgz. Pollack.et «Z. (l965e,b) described a staining procedure to localize enzymes in the¥mycoplasma,orgeniam. Rottems and gesin.(l966) localized adenosine triphosphatase activity in the mycoplasme_membrane_ by a colorimetric method. Adenosine triphoephatase participates in the: utilisation of energy to transport nutrients from the media across the membrane; ‘Munkres and Hachte1,(l967) localized acid and alkaline phoe— phatase inside the organism associated with the metabolism of phoepholipids.‘ Patholggy. The changes in tissues associated with the invasion of myco- plasmiwere described before the causativeagent was isolated. An extens- ive, detailed reportfdescribing the pathogenesis of ITS by light microsc0py was published by Jungherr (1948). He-also described the-air secculitie 8 end.pneumonie which often accompany.the sinusitis. Hitchner (1949) described the histepathology in birds of different ages-and duration of infection; Berber (1962) traced the progressive development of lympho- follicular nodules first described by‘Nelson-(1935) and assumed by Jungherr. (1949) to be pathognomenic for ITS. Gordy and Adler (1957) isolated myoo—- plasmas from the,brains of turkeys and believed them to be the cause of. encephalitis. -II9‘t‘.95:9§é_9995391‘ Many antibiotics have been employed in eggs and birds to treat CRD. Aureomycin.(Leece and Sperling,.1953), chloromycetin, streptoomyin, tetracycline (Domermuth, 1958) end terramycin.(Leoce and Sperling,=l955: Yemenoto.and Adler, 1956) were:found to be effectivea Benton and cover (1958) and Domermuth (1958) found nitrofuren compounds~y effective, Sulfonemides have been found effective .onlylin‘thle egg- (won; and James, 1953). Gale at al. (1967) found tylosin effective: against combinations of infections.. The addition of sodium sulfate to_ the water as e drug potentiator_increeses the blood level of chlortetree cycline. Gale and Beughn (1964) found that this increase in the blood level did in fact increase the therapeutic‘effectiveness of chlbrtetreq cycline against.erperimentel,mycoplaeme infections. The.firit effective treatment for 029 was.gx silver.nitrate described by Tysser,(1926) when he,first recognisdd the disease in the.United States. “Seams? . 9% mrsnfiécmsm . The construction_of the first crude light.microscope'in,the.16th century revealed.new and_mnexplored worlds. Although these developments~ opened a vast field of.exploration to the.scieetist,‘the factor restrict— ing the study of the,ultrsetructure‘offcells and.tiseuee was the funde- mental nature of light,-which imposes a physical.limit to the resolution. 9 This problem was partially alleviated in the 20th century by the deve10p— ment of workable electron microsc0pes. The biologist has now been given visual access to worlds between the cellular and molecular levels. This new vision spans 3 orders of' magnitude (10'4 to 10‘7 cm) which, when explored, must be interpreted and correlated with findings of light microsc0py, gross examination of the entire organism, and biochemistry. The light microscepe reached the peak of its deve10pment more than 2 centuries after its invention; however, the electron microscoPe reached a comparable level in the short span of 2 decades( In the past 50 years it has been possible to examine and evaluate a vast cellular and histo— logical realm by light micrcscOpe. It will take a far greater time to record and interpret the information made visible on the ultrastructural level. Theoretical. Principles of physics used in today's electron microscOpes were understood in the latter part of the 19th century. However, it was not until de Broglie's theory (1924) that moving electrons can be assigned very short wave lengths and Busch (1926) demonSErated that suitably shaped magnetic or electrostatic fields could be used as true lenses for an electron beam, that satisfactory progress was made in constructing a functional electron microscope. Ruedenberg applied for the first patents on an electron microscope in which the principles of physics and electronics known at that time were incorporated. As it turned out, the instrument was more descriptive than functional, but the evolution of electron microscOpy had begun (Wischnitzer, 1962)o lO Beuche and Johnson (1932) built an electrostatic instrum.e nt which was designed to produce an enlargement of electron emitting source. The prototype microscope incorporating the principles currently used in design was builtb by Knoll and Ruska (1932). Their instrument utilized an electron transmission gun which produced e; ’ec tron phot wmicrographs of an enlarged illuminate specimen (Ruska, 1932)o Driest and Muller (1935), using Ruska's instrument, first demonstrated resolving powers greater than the light microsc-Ope° Von Berries and Rusaka (1939) constructed an electron microscOpe, capable of resolving 100 K, which was built and marketed on a commercial basis in 1939. Sophis- tication cf the instrument was rapid. Von Ardenne (1944) achieved resolvu o 7, . ° . ing powers of 12—15 A and Hillier (i9fi6) demonstrated 10 A resolVing T) powero Hillier and Ramberg (1947) develeed the compensating objec try that improved image quality by eliminating astigmatism in imperfect mag— netic lenses. (3 In the span of 20 yea s the scion e of electron microscopy has been refined to a point that today‘s microscopes possess esolution capabili- ties which border on theoretical limits, Staining Uranyl acetatel Watson (1958) was the first investigator to explore the use of uranyl acetate as an electrtn stain: It was at first considered I‘m a stain with little speci'ic' eca as proteins will stain fairly H I"? v; 0‘ intensely with uranyl acetate but cytomembranes do not stain as well” It does not produce the vivid pi :tu res that lead does. Huxley and Zubay €l961) have demonstrated that DNA binds a Specific amount of uranyl acetate (agpfl.ximately equivalent to its own dry weight) Marinozzi and Gautier (1962) also emphasized the specific binding of ll uranyl acetate with nucleoprotern, the exact character of the reaction depending in part upon the fixation employed. It is now generally agreed that uranyl acetate is a specific DNA nucleoprotein stains Lead. An interpretation of alkaline lead differential staining was made by Marinozzi (1963)° He concluded that lead reacts with reduced com- pounds such as osmium which may be present in the sections, Lead is bound specifically to RNA in the section by Other reactionso Thus, after osmium tetroxide fixation, alkaline lead compounds act as a general stain, whereas after formalin fixation they become RNA specific stains. Daems and Persijn (1963) have postulated at least 3 mechanisms of attachment of lead to reactive groups. Firsr, lead is attached to cyto- membranes; this reaction requires the presence of negatively charged reduced osmium on the polar groups of the phosphatideso Second, staining of glycogen is based on the chelation of lead by hydroxyl groups of car— bohydrateso Finally, nucleoprotein staining has a preference for RNAO The combination of uranyl acetate and lead hydroxide is commonly used today to stain aldehyde—fixed tissuesa Histoihemistry of Turkey Sinus There is a vast amount of work published on the histochemical reac— tion of various tissues and cellsr Pearse (1958) described some of the applications of histochemistry in cellular pathology; There is no litera~ ture known to this author concerning the enzyme histochemistry of the normal or infected turkey sinus, In an attempt to anticipate what could be expected to happen in the turkey sinus because of myCOplasma infection, similar investigative work in mammals was reviewed) In the presence of an infECtiOn with Mycobaoterium paratuberculosis, Merkel at aZg (1968) reported an increased activity of alkaline 12 phosphatase in the tissue cells, while lesions and bacillary products caused an increase in acid phosphatase and esterase activity in the macrOphages. The enzymatic response in tumors may be dependent on size of growth as shown by Loeb (1965), who studied the intensities of 4 enzyme—catalyzed reactions in 56 neOplastic tissues of the dog. He found that intensities of.reactions in neOplastic tissue were in fairly good agreement with enzyme-catalyzed reactions in their tissues of origin. In addition, intensities of the 4 enzyme~catalyzed reactions were highest in small tumors and lowest in large tumors. The mean value of tissue lactic dehydrogenase activity was significantly higher in malignant tumors than in benign tumors. This was thought to be from an increased synthesis of the enzyme by the tumor. A second explanation not mentioned by Loeb cannot be ruled out, which is that storage of the enzyme in malignant tissues may be enhanced as a result of autolytic changes found in neo— plastic tissues. A high activity of tumor tissue phosphohexose isomerase correlated closely with the prOperty of invasiveness of tumors. Alkaline phosphatase was higher in ssteogenic sarcomas than in other neOplasms. Activities of 5 lysosomal enzymes were investigated in 4- to 6—week— old normal lambs and lambs suffering from white muscle disease which results in tissue necrosis (Desai, 1966). In all 1nscances, enzyme activity was higher in dystrOphic muscle than in normal muscle with the increase being 35-fold for aryl sulphatase, Sufold for B—galactosidase and cathepsin, and 2-fold for acid phosphatase. flydrolytic enzymes. This group of enzymes includes esterases that cata- lyze the cleavage of phOSphoric acid esters. 13 Alkaline phosphatase. PhOSphate esters, such as glyceroph05phate, glucose-l-phosphate, creatinine phOSphate, and the nucleotide of adenosine triphosphate, are split by alkaline phOSphate activity (Gomori, 1949; Feigin and Wolfe, 1955; Friedenwald and Gryler, 1958). Alkaline phospha- tases hydrolyze most orthOphoSphomonoesters at an optimum pH of 9.0 to 9.6. Many authors feel alkaline phOSphatase is composed of several enzymes which are active upon closely related substrates with Optimal activity at alkaline pH (Emmel, 1950; Burgos at al., 1954, 1955). The function of both acid and alkaline phosphate is the dephosphorylation necessary for absorption, transport and metabolic control. They may be active in some ester synthesis and maintenance of intracellular inorganic phosphate suf— ficient for osteogenesis (Morton, 1961). There are many reports of alkaline phosphatase localized in epithelial lining cells of mammals (Shuitka, 1960; Padukula at al., 1961; Pearse and Riecken, 1967; Maronpot and Whitehair, 1967; Thake, 1968). Acid phosphatase. There are several enzymes in this group. Phos— phomonoesterase II and nonspecific acid phosphatase hydrolyze several phosphoric acid esters at an optimum pH of 5.0 (Barka, 1960; Barka, 1961). Acid phosphatases are widely distributed in animal tissuesc Gutman (1938, 1940, 1941) extensively studied the prostatic acid phOSphatase and proposed possible functions. Until recently, however, histochemists have tended to overlook the importance of this group of enzymes. Fennell (1966) reported a marked increase in acid phosphatase in chicken cells undergoing necrosis from tissue graft rejection. Burstone (1961) developed a cytochemical method which couples alpha- naphthal Azo dye to demonstrate hydrolysis of phosphoamideso Improvements of this method are widely used today. 14 Proteolytic enzymes. Proteolytic enzymes have 2 major subgroups, the pro— teases and the peptidases. The protease subgroup hydrolyzes peptide bonds between Specific amino acid combinations in the protein chain. Peptidases hydrolyze only the terminal bonds of the protein chain. They are specific for either —n— or -c— terminal bondings. Leucine amingpeptidase. This enzyme is a peptidase (exopeptidase) which requires an n— terminal free a-amino acid group for its action. The aminOpeptidase acts most rapdily on terminal leucine residues but also liberates all other amino acids found in proteins, although its release of certain residues is very slow. The enzyme is widely distributed in animal cells that have a rapid protein turnover (Ticktin and Trujillo, 1966). It is specifically located at the level of the microvilli in the small intestine of man (Pearse and Riecken, 1967). Nachlas et al. (1957a) and Nachlas at al. (1962) described a tissue localization staining procedure that is still widely used. He also suggested the use of small intestine as a control. Oxidative enzymes The dehydrogenases. Most of the dehydrogenase enzymes commonly studied are in the tricarboxylic acid cycle. This is the anaerobic part of the coupled respiration of the cell. Products of glycolysis progres— sively dehydrogenated with carbon fragments going into other pathways for nutrient synthesis and energy are generated. The hydrogen, in a reduced form, is removed through a series of cytochrome enzymes. It is then reacted with oxygen to form water and in the process more energy (ATP) is formed. Pasteur recognized that glycolysis was linked to res- piration of the cell in his early biochemical work. 15 Succinic acid dehydrogenase. This enzyme catalyzes the conver- sion of succinate to fumerate in the tricarboxylic acid cycle (Krebs cycle). The hydrogen ion is transported to the reduced flavine adenine dinucleotide as its first step in the electron tranSport system. Nachlas et al. (1957b) reported high activity of many tissues in the mammal, including skeletal muscle. Succinic acid dehydrogenase is not a soluble enzyme and is always found associated with mitochondria. Isocitric acid dehydrogenase. This is a tricarboxylic acid cycle enzyme that catalyzes the reaction of isocitric acid.to alpha keto- glutaric acid through the rapid intermediate compound oxalosuccinic acidc There are 2 products of this reaction. Carbon dioxide is lost from the carbon chain and hydrogen ion is given up to the electron transport system with the yield of one ATP. Hess at al. (1958) described the localiza- tion procedure with skeletal muscle as the control tissue. Malic acid dehydrogenase. The reaction, malic acid oxidation to oxaloacetic acid in the presence of malic acid dehydrogenase and DPN, is found in the mitochondria. This enzyme is insoluble and has never been isolated free of the mitochondria. Nachlas (1957) reported a localization procedure using kidney as a control tissue. Pearse and MacPherson (1958) reported general activity in mammalian tissue cells with high activity in the kidney. Lactic acid dehydrogenase. Henderson (1965) suggested that the lactic acid dehydrogenase may regulate the reaction between oxidized and reduced diphosphOpyridine nucleotide. Isoenzyme LDHS may react with DPNH to convert pyruvate to lactate and DPN whereas LDHl interacts with DPN to convert lactate to pyruvate and DPNH. Nachlas (1957) localized 16 the enzyme in many tissues of the body. He used smooth muscle for a con— trol tissue. It yields a constant reaction. Alcohol dehydrogenase. This enzyme is widely distributed in both animals and plants. In a review of the Optic system Pirie(l958) describes the function of an alcohol dehydrogenase in utilization of vitamin A in the visual system. By Hess' procedure (Hess, 1958) concentrations of alcohol dehydrogen- ase can be localized in epithelial cells. Glutamic acid dehydrogenase. This enzyme catalyzes the combi- nation alpharketoglutaric plus hydrogen plus ammonia ion in presence of DPNH to form L-glutamate. Thus glutamic acid, an amino acid, is formed frmm a product of the TCA cycle. Glutamic acid dehydrogenase is almost universally distributed in animal tissues. Steroids will cause the disso- ciation of glutamic acid dehydrogenase and may limit its activity in the body (Yielding and Tompkins, 1962). Glucose-6+phosphate dehydrogenase. G1ucose-6-phosphate dehy- drogenase catalyzes the first reaction in.the pentase monOphosphate shunt-- glucose 6 phosphate to 6—phosphog1uconolactone in the presence of TPN. Because the equilibrium constant for this reaction is large, it has often been used to generate TPNH (Green, 1960). This enzyme is localized by the procedure of Hess (1958) in many tissues. Esterases Myristrol cholinesterase. This enzyme catalyzes the breakdown of myristrol choline at neuromuscular junctions. Myristrol choline is the specific substrate used to study the cholinesterase distribution in 17 nerve tissues of dogs (Hard and Peterson, 1950). The myristrol method was first developed by Gomori (1948). Holmstedt (1957) modified and refined the procedure. Duffy at al. (1967) described a procedure to demonstrate cholinesterase adaptable for both cytochemistry and electron microscOpy. MATERIALS AND METHODS Electron MicroscoPy EXQerimental animals. Day-old, white broad-breasted male turkey poults were obtained from a certified mycoplasma—free flock. The first 2 weeks the turkeys were housed in electric battery brooders, then transferred to the floor. The birds were divided into experimental and control groups and housed in separate isolation rooms, where feed and water were available ad Zibitum. Movement of other animals was minimal in the building, and special care was taken in handling the birds to prevent infection. At 4 weeks of age, the birds in the experimental group were inocu- lated into the infraorbital sinus with 0.2 m1. of a newly resuspended broth culture of 1y0philized MchPZasma gallisepticum (S-6 culture #1299 gl(Pl), obtained from Dr. Yamamoto, Davis, California). An excess of birds was inoculated to assure adequate numbers of infected birds at each sampling period. One week through 6 weeks after the inoculation date, 2 clinically affected turkeys and 2 control turkeys were killed each week (Figures 1 and 2). Mucus was withdrawn from each infected bird and cultured on agar-base media and in broth (see Appendix A) to confirm that myc0p1asma was the causative agent of the sinusitis. Just prior to killing the turkeys, each sinus was injected with 4% neutral (pH 7.2) filtered solu- tion of glutaraldehyde. Immediately after the head was removed, each outer sinus wall (Figure 3), including the skin and mucous membrane, was 18 l9 dissected free, pinned flat to paraffin in a petri dish with the mucous membrane uppermost, and flooded with a quantity of glutaraldehyde suffi- cient to cover the tissue. After 4 hours of fixation the glutaraldehyde was decanted and replaced by Sorensen's buffer (pH 7.2), which was changed 6 times in.the next 2 hours. The tissues thus preserved were stored in Sorensen's buffer for a maximum of 1 month (see_Appendix B). The tissues were prepared for embedding by blotting dry with a paper towel and positioned under a dissecting microscOpe. With an oil—free, singleeedge razor blade the skin and mucous membrane were separated through the loose connective tissue of the submucosa of the sinus (Figure 4). The membrane was divided into halves and a 3 mm. strip was sliced off. This was diced into 2 x 3 mm. sections. These pieces were postfixed in 12 osmium tetroxide, dehydrated, and embedded in epon 812* using rubber ,flat molds (see Appendix C). The heat-cured blocks were mounted in a flat embedding chuck** (vise- type) and trimmed by hand with a razor blade according to Sorvall's tech- nique (Thin Sectioning, p. 61) to a 1—mm.-wide trapezoid-shaped block face. Microtomy. The sections were cut on a Sorvall MT—Z Porter-Blum ultra- microtome. This instrument utilizes a double pivot cantilever arm to determine the advance. The specimen, which is mounted in a vise-type‘ holder, passes over the knife edge and the section is floated on water. The speed of the cantilever arm does not determine the section thickness. Glass knives were used to cut the sections. These knives were pre- pared fresh each day. They were broken on an LKB 78003 Knifemaker from precut strips of LKB glass.*** *Ladd Research Industries, Inc., Burlington, Vermont. **Sorvall Corporation, Norwich, Conn. ***LKB-Produkter Ab., Stockholm, Sweden. 20 As a preliminary screening of blocks to assure prOper orientation of tissue, "thick" sections (approx. 211) were cut and stained (see Appendix D). The blocks, on which cross sections of the Ultra thin sections were out according to the procedure in the "thin section" guide (Sorvall). The sections were floated on acetone-water in a boat around the glass knife made of black plastic electrician's tape.* Silver to gray sections were selected which were between 60 and 90 m1 in thickness. A color scale chart, "continuous interference color and thick- ness scale for thin sections" (Sorvall), was used to make the thickness determinations. The thin sections were picked up on the dull side of a 400-mesh c0pper grid (LKB). The grid was then dried on filter paper and stored in LKB grid boxes under vacuum. Staining. Reynolds (1963) lead citrate was_the primary stain with uranyl acetate as a counter stain (Spink, 1968) (see Appendix D). The grids were floated tissue-side down on drops of lead citrate stain in porcelain depression dishes. After 15 minutes they were blotted dry on filter paper and transferred to draps of fresh, saturated uranyl. acetate solution. After a l-hour staining period, the grids were washed with distilled water and a jet of 0.04 N NaOH, dried, then stored under vacuum. Micrdscopy. The tissues were examined under an RCA EMU-4 electron micro- scope"** This instrument has.a resolving capability of 8 A and a useful magnification power of 1,400-200,000 times. With.the "selected area. '*The 3-M Company, Minneapolis, Minn. **RCA Scientific Instruments Engineering, Camden, N.J. 21 defraction" feature of the instrument, the grids were scanned at a 1:1 ratio to locate the tissue sections. The instrument is capable Of 50 and 100 Kv. Operation of the gun assembly. Fifty Kilovolts were most suited for this work. The condenser lens system located just under the gun assembly controls the intensity Of electron beam on the specimen. The specimen chamber is deSigned for minimum temperature variation and minimum contamination of the column. Objective lenses are coil-heated and water-cooled for constant Operating temperature. The projector system, which includes the intermediate and projector lenses, forms the final image. The image is projected on a fluorescent.viewing screen. Laminated lead glass shields the Operator from x-ray exposure. Photggraphy. Kodak Electron Image plates 3-1/4 x 4 inches were used. The EMU-4 Electron Microscope has a capacity of 6 racks of 3 plates each. A beam intensity meter and an automatic exposure timer are built into the instrument. Two exposures were made of each field at 2 and 4 seconds with a beam intensity of 0.5 electron speed. After exposure the plates were removed from the instrument and pro- cessed according to Kodak procedures (see Appendix E). Printing. The 3-1/4 x 4-inch plate negatives were printed using a tungsten lamp enlarger. An IlfOprinter* was used to make a scanning proof of each plate. The final prints were produced by the pan method using Dektol** developer, Kodak stop bath--with indicator (10 seconds), Kodak fixer (10 minutes), and Kodak Photoflo in the wash-(20 minutes). The prints were dried on a drum print dryer. *Ilford Company, Essex, England. **Kodak, Rochester, New York. 22 Histochemistry Experimental animals. Day-Old broad-breasted white male turkeys were divided into 2 groups and housed in separate heated isolation rooms with feed and water available at all times. Mycoplasma gallisepticum, S-6 Strain (courtesy Of Dr. Yamamoto, University of California, Davis, Cali— fornia), was injected into the sinuses of experimental turkeys at 4 weeks of age. The other group was held as controls. One week later and for 5 successive weeks, 2 infected and 2 control birds were killed each week and the sinus membranes were stained for activity Of 11 selected enzymes. Experimental design. The entire experimental procedure was repeated twice. This involved 2 different groups Of birds, but all birds were the same age for any given point in the experiment and all birds were from the same myCOplasma-free parent stock. Enough birds were infected to assure' that infected birds would be available each week. Only birds with swol- len sinuses were selected and at necropsy the sinus cavities were examined for mucous exudate and cultures were taken to confirm myCOplasma infection. MQCOpZasma gallisepticum was recovered in pure culture from each bird. Tissue sections and histochemical techniques. The sinus wall, including skin and mucous membrane, was removed and immediately frozen on steel specimen holders that were partially submerged in acetone and dry ice. The frozen tissues were then held at —30 C. in the storage chamber of a Pearse cold microtome (cryostat), Type H.* Sections were cut at 12 u. transferred to coverslips and incubated for 30 minutes. The staining procedures were started less than 2 hours *South London Electrical Equipment CO., London. 23 after the turkeys were killed. Procedures for localization of 11 differ- ent enzymes which can be grouped according to the type of reaction cata— lyzed were applied to individual sections. Localization of alkaline (AP) and acid phosphatase (acid P) were determined by the procedure Of Burstone (1961), leucine aminOpeptidase (LAP) by the method of Nachlas et al. (1957a, 1962), succinic acid dehydrogenase (succinic DH), malic acid dehydrogenase (MDH), and lactic acid dehydrogenase (LDH) by the method of Nachlas et al. (1957b), isocitric acid dehydrogenase (ICDH), alcohol dehydrogenase (ADH), glutamic acid dehydrogenase (GIDH), and g1ucose-6-phosphate dehydrogenase (G6PDH) according to Hess et a1. (1958), and myristrol cholinesterase (Che) according to Gomori (1949). Intensity of enzyme staining for a positive reaction was given a value Of 1+ rather than a graded score because the intensity Of enzyme reaction under these conditions appeared maximal in both control and experimental tissues (Table 1). Controls. Sinus tissue was taken from uninfected control turkeys of the same age as the infected turkeys for simultaneous staining. In addition, a tissue known to have a high concentration Of each enzyme was taken from a control bird and stained simultaneously as a positive control for the histochemical procedure as well as for comparison with the infected sinus tissue. These positive control tissues were: liver for G6PDH, Che, GIDH, and ADH; kidney for MDH and AP; skeletal muscle for succinic DH and ICDH; small intestine for LAP; smooth muscle for LDH; and thymus for acid P. A final control was the incubation of a section of uninfected sinus with- out the specific substrates (Table 1). Table 1 . 24 for eleven enzymes"‘ Enzyme, reaction catalyzed, control tissues and references Reaction Control Enzyme Catalyzed Tissues References Alkaline phosphatase Nonspecific phos- Kidney Burstone, phomonoesterase 1961 pH 9 Acid phosphatase Nonspecific phos- Thymus Burstone, phomonoesterase 1961 pH 5 Leucine amino- Hydrolysis of Small intestine Nachlas peptidase N—terminal at aZ., 1957a peptide bonds Nachlas et al., 1962 Succinic acid Succinic acid 3- Skeletal muscle Nachlas dehydrogenase furraric acid et al., 1957b Isocitric acid Isocitric acid 1- Skeletal muscle Hess at aZ.,‘ dehydrogenase c-ketoglutaric acid 1958 Malic acid Malic acid": Kidney Nachlas - dehydrogenase oxalocetic at al., 1957b Lactic acid Pyruvic acid 1- Smooth muscle Nachlas dehydrogenase lactic acid at aZ., 1957b Alcohol dehy- Acetaldehyde'i Liver Hess et al., drogenase alcohol or 1958 alcohol +02, Glutamic acid c-ketoglutaric acid't- Liver Hess et al., dehydrogenase glutamic acid 1958 Glucose-6-phosphate dehydrogenase Myristrol cholinesterase G1ucose-6—phosphate . phosphoglucono lactone Hydrolysis Of fatty acids 1- Liver Liver Hess at al., 1958 Gomori, 1948 RESULTS Electron Microscopy» Sections from each week, 1 through 6, were examined. The change in the pattern of infection each week was not significantly different, so the weeks, 2 through 5, have been deleted from the study and a comparison of Wéeks 1 and 6 will be reported. The electron microscOpe examination of sinus tissue after 1 week of infection revealed myCOplasma organisms packed between and within the cells (Figures 5 and 6). Even though organisms were closely packed, they were not distorted from pressure. They appeared as round bodies, each with a dense dark central structure surrounded by a narrow lighter zone containing granular particles. Granule-filled spaces and clear spaces with no organisms were also seen (Figure 7). There was slight variation in size among the organisms but nO pleomorphism. Intracellular populations of mchplasmas varied from large numbers, densely packed (Figure 6) to as few as a single organism in some cells (Figure 8). Mycoplasmas were located in the cytOplasm but never in the nucleus. They appeared as deep in the cell as the level of the nucleus but never below it (Figures 8 and 9). The nuclei of infected cells were intact, and cells remained attached to the basement membrane (Figure 9). Many ciliated epithelial cells also had microvilli (Figures 10 and 13). In heavily infected cells both of these structures were reduced in number or appeared to be absent (Figures 11 and 12). In lightly infected cells (Figure 8) these structures remained unchanged. In infected cells 25 26 which had lost most of their microvilli, the microvilli that remained appeared as short rounded projections. Any uininfected cells that had both fully developed cilia and microvilli appeared normal even though they were adjacent to infected cells (Figures 12 and 18). Internal.ce11ular structures, other than the nucleus, were not readily visible in cells that were filled with organisms (Figure 6). In cells containing few organisms (Figure 8) and in uninfected cells, the internal structures were only mildly affected. In Figure 11 there is some swelling of the mitochondria. There also was some increased granularity of the cytOplasm in some cells from infected sinuses but the cells and intra- cellular structures appeared tO be morphologically normal (Figures 12 and 14). The contour of the epithelial surfaces in infected sinuses was irregu- lar (Figures 5, 11 and 14) compared with the even, symmetrical surfaces of membranes from control birds (Figure 13). Occasionally, however, unin— fected cells lying between 2 infected cells were compressed and bulged slightly above the surface (Figure 18). In parasitized cells that were undergoing degeneration, only the distal part of the cell seemed to be affected. This segment of the cell and its contents were sloughing away from the base of the cell (Figure 15). In spite of these degenerative changes, the nuclei appeared intact and the cytOplasm between the nucleus and basement membrane was normal in appearance. There was an occasional cell that had some Of the early stages of necrosis (Figure 15). Some exudate was lying on the free surface of the cells (Figure 15). This was probably mucus and cellular debris. Mycoplasmas were seen in the exudate but not in the large numbers found between or within cells. 27 At the sixth week, few mycoplasmas were seen but many bacteria (Pbeudbmonas 3p.) as well as fungal spores had appeared. In some cells at this stage, regardless of the infectious agent, the microvilli were reduced in number and shortened, and the contour Of the membrane surface was still irregular (Figure 14). At this time, however, some cells appeared to be regenerating in spite of the infection. Mycoplasma organ- isms were not found in the cells, and relatively few were seen in the exudate that filled the sinuses. In some areas there appeared to be a healed surface covered by healthy-appearing ciliated cells (Figure 17). Histochemistry Clinically, slight swelling Of the sinuses was noticeable 96 hours after inoculation (Figures 1 and 2). After 1 week sinusitis was apparent, and after 6 weeks the sinuses were enlarged, soft, and fluctuating; and. mucous exudate appeared in the nostrils if they were pressed. Mchplasmas were readily cultured from the exudate. . Four of the 11 enzymes investigated were present in sufficient con- centrations in the epithelial cells to be localized by the staining methods used. These 4 (succinic DH, ICDH, MDH and LDH), all members of the dehydrogenase group, are illustrated in Figures 19, 20, 21 and 22. Furthermore, the intensities of the enzyme-catalyzed reactions were essentially the same in the epithelial cells of both experimental and control birds. The 7 other enzymes were absent or in concentrations insufficient to permit their localization following 30—minute incubation in substrate solution. MyCOplasma infection did not cause the appearance of any enzymes not found in the control cells; neither did it cause any to disappear. Thus, in the presence of infection, there was no apparent change in the 28 qualitative composition of the cellular enzyme components. In the third and sixth weeks, slight-succinic DH and MDH activities were found outside of the cell at the free surface. In the fifth week of infection in a 9-week-Old bird, AP was found in the submucosa. It appeared as a diffuse reddish-stained substance evenly distributed in the tissue, and numerous localized areas of AP activity were seen in the capillary walls. Alkaline phosphatase had not been noted in these locations before. All of the tissues selected for controls because of known high levels \. of given enzyme activity were positive in these procedures (Table 2). 29 Figure 1. Front view of.a turkey infected with Mycoplasma galliseptioum S-6. Swollen sinus and nasal discharge are clinical signs of infectious turkey sinusitis. Figure 2. Side view Of a turkey infected with Mycoplasma gallisepticum S-6. 30 Figure 3. Outside surface of sinus skin covering. Figure 4. Inside surface Of sinus and mucous membrane lining. Arrow indicates area sampled for electron microscOpy. 31' I... 9. «3...». . Myoop Zasma gallisep tioum Infected turkey--first week. packed between cells (arrow). x 6,800. Figure 5. 32 MyCOplasma filling the Infected turkey-—first week. cytOplasmic space of the epithelial cell with loss Of fine structure. The nuclei are depressed toward the cell base (arrows). x 13,600. Figure 6. 33 Figure 7. Infected turkey--first week. The organisms appear as closely packed, round, structureless bodies surrounded by a narrow zone of granular particles (arrow). Organisms have disappeared from clear spaces (8). There was a slight.variation in size among the organisms but no pleomorphism. x 20,400° 34 .3.-.h2 . . ..... . . . .. “1.44.”... . . . ‘1. I 0 . Q Q h . a . .. ... . . .... .- . _. ...... .4 b . A. . .... pg m,ee ......t. ‘J' I H~II 13% 4 PT 1: fl . f . ,.‘ .. a .I‘lr v a ... n J: ..v 1.. ‘nl. r J . ; . . m - A single organism at the Infected turkey--first week. Figure 8. level of the nucleus (arrow). The cell retained its fine structure and An adjacent cell is more severely infected and has lost surface and cytOplasmic structures. x 9,520. cilia. 35 :ct‘f" ' Fag; Figure 9. Infected turkey--first week. Basal portion of the cells at the attachment to the basement membrane appear normal. x 4740. x- . III-,1... x 2.41-1.14 36 Figure 10. Infected turkey--first week. There is no evidence of Mycoplasma galliseptioum infection in this section. Normal mito— chondria (Mi), basal granule Of the cilia (Bg), and microvilli projec- tion (Mv) are seen in this cell. x 6800. 37 Figure 11. Infected turkey-—first week. Infected cell (center) between 2 normal cells. Mitochondria (M1) and cilia (C) are seen in the normal cells but not in the infected cell. The cellular interdigi— tations appear normal (Id). x 13,600. 38 w M ... \'\ \\ :.°|.: Figure 12. Infected turkey--first week. Loss of cilia and micro- villi from infected cells. Normal appearing fine structure in unaf- fected cell. x 6800. Figure 13. Control turkey-—first week. Even contour of the surface cells. There are normal appearing fine structures with the nuclei at different levels in the cells. x 6800. 40 Contour Of the cells is irregular with the loss of cilia and shortened microvilli (Mv). x 9520. Infected turkey--sixth week. Figure 14. -\.. Figure 15. Infected turkey--first week. Most nuclei appear normal; however, some show signs Of early necrosis (arrow). There is degenera- tion of the cytOplasm of the cells. x 6800. 42 Figure 16. Infected turkey--sixth week. This cell has increased ribosomes (R1) and nucleolus (Nu) which indicate active protein synthe- sis. x 20,400. 43‘ Increased size and number Figure 17. of mitochondria indicate higher cellular metabolic rate through the tri— Infected turkey--sixth week. Lysosomes (Ly) and Golgi apparatus (61) are also carboxylic acid cycle. x 6400. seen o t )r- l Normal cell is compressed Infected turkey--first week. Figure 18. between 2 infected cells. x 6800. 45 Figure 19. Succinic acid dehydrogenase. The enzyme is localized in the free surface of the cell. x 550. Figure 20. Isocitric acid dehydrogenase. Enzyme activity distri- buted throughout the cytoplasm. x 550. 46 Figure 19 Figure 20 47 t Figure 21. Malic acid dehydrogenase. The enzyme is concentrated i in the free surface Of the epithelial cell. Figure 22. Lactic acid dehydrogenase. The enzyme activity is evenly distributed throughout the cytOplasm. 48 Figure 21 ' Figure 22 49 cowuoom canoufioma: a 0 Hammond mua>fiuom a + >uH>Huoo on n I msaHm cocoouaH I H« modem Houucoo I we I I I I I I + + + + + + + + I I I I I I I I 0 OH I I I I I I o + I o + + + o I I I I I I I I m m I I I I I I + + + + + + + + I I I I I o I I a m I I I o I I + + + + + o + + I I I m I . I I I m n I o I o I I + + + o + o + + I I I I I I I I m o I I I o I I + + + o + o + + I I I o I o I I H n H o H o H o H u H o m, c H o H o H u H o «H so aoHu A.esv cameo mHHe- wee-) meH mm: mouH me maH emu m ma qucaH «we UHcHu eHua Ho .xz loom madam Houanuooumsa hexane ca oaaoo Hoaaocuaeo vouoomsHImamoHoooxa one HOHHGOO mo hua>HuOo camucm . N canon. DISCUSSION Electron Microscopy Most Of the mchplasmas, after 1 week of infection, were situated either between the epithelial cells or within them. Very few were lying free in the lumen or trapped in the exudate. Since some cells were filled with organisms or spaces between cells were packed, it must be that a characteristic of this strain is to colonize in certain areas rather than being distributed diffusely throughout the lumen and tissue. The morphologic appearance of the myCOplasmas in these tissues resembled the forms that have been seen in human tissue. The large,‘ round, dense body is the myCOplasma, and the granular particles that sur- round and cling to the organism are probably only proteinaceous material. The light Space is probably a vacuole in which the organism lies (Figure 7). The myCOplasmas reported by Dmochowski et a1. (1967) and Hummler and Armstrong (1967) were also in vacuoles in the cells. The granule-filled spaces devoid of owganisms and the empty clear spaces were probably where organisms had undergone degeneration or had dropped out during sectioning. Mchplasmas did not appear flattened or distorted, in spite of their densely packed arrangement between and within cells. Since these organisms occupy space, it must be that the cell changes shape to accommodate the organisms. One week after infection, even with large numbers of organisms pres— ent, only a few were undergoing degeneration. This may not be an accurate 50 51 observation, however, since markedly degenerate organisms might not be readily recognized and only those with some form remaining would be seen. Since numerous organisms were packed into the cells, it was diffi— cult to evaluate the fine structure of the cytOplasm. Figure 7 shows no evidence of mitochondria admixed with the mycoplasma. The mitochondria in some of the cells adjacent to infected cells appear to be unaltered in size, number or structure, while others (Figure 15) show structural changes consistent with increase in the tricarboxylic acid cycle enzymes. As stated in the section on histochemistry, however, we measured no increase of TCA cycle enzymes by our methods. Bacteria were not part of the pOpulation of the sinus the first week. At the sixth week, however, bacteria and fungal spores were pres- ent but fewer myCOplasmas were seen. Microvilli on infected cell sur- faces were decreased in number and were shorter than similar structures on normal cells. The shorter microvilli on cells after 6 weeks of infec- tion may indicate early recovery and regeneration of these structures rather than their continuing degeneration. It appears that cilia and microvilli are affected by any type of infection, since,_at 6 weeks, with few myCOplasmas remaining, these structures were still not normal on some cells. The loss of surface structures in the presence Of infection is not unique with these cells. Microvilli on jejunal epithelium.were also fewer in number and shorter than normal in the presence of bacterial or viral infections. In germfree baby pigs infected with the virus of trans- missible gastroenteritis, the villi of the jejunum were lost for a time (Trapp at aZ., 1966). During the infection, their position was indicated only by a low, broad stump with wide spacing between these bases. Thake (1968) also reported that microvilli on epithelial cell surfaces in pigs 52 infected with the same virus were markedly reduced in number and were much shorter than normal. In germfree baby pigs given virulent Escherichia coli organisms orally, intercellular spaces were widened, microvilli on the epithelial cells were shortened and reduced in number, the terminal web was vacuolate, cytOplasm was pressed to the periphery Of the cells by large vacuoles, and cells became degenerate. Bacteria invaded the cells and were present in the cytOplasm (Drees, 1969). In myCOplasma infection only the distal part Of some cells was degen— erating but it appeared that, unless further degeneration occurred, the cells would readily recover if the infection were removed. This is sug- gested by the fact that infected cells remained attached to the basement membrane and the nuclei were generally intact. This limited peripheral degeneration of cells is in agreement with Observations that necrosis of the cells in the early stages of infection is minimal (Jungherr, 1949) and with the absence of acid phosphatase in the infected cells. There were occasional cells (Figure 15) with wrinkled nuclear membranes and loss of rough endOplasmic reticulum. These may be examples of early necrosis as described by Sandritter and Wartman (1967). There was no other evi- dence of necrosis seen such as karyorrhexis or karyolysis. Bacteria in the sinus of these birds 6 weeks after infection con- firms the work Of Groupé et a1. (1948). Perhaps some of the profuse pro- duction of mucus in sinusitis may be caused by secondary infection rather than solely from M. gallisepticum, since they were less numerous after 6 weeks of infection than were bacteria and fungal spores. The decreased pOpulation of organisms after 6 weeks of infection suggests that some resistance may have developed in the host. This decrease of organisms in later stages of infection and the appearance 53 of bacteria and fungal spores have also been reported by other workers (Lutsky and Organich, 1966; Organich at al., 1966; White et aZ., 1954). Also, healing Of cells apparently progresses rapidly at this time, as indicated by the increase in rough endoplasmic reticulum for protein synthesis (Figure 16). The mitochondria of recovered cells (Figure 17) are increased in size and number. This also indicates an increase in cellular metabolic activity through the TCA cycle enzymes. There are also more lysosomes seen in the healed cells, which indicates increased hydrolytic enzyme activity. It would be expected that with such increase in intercellular enzymatic activity, some changes in the histochemistry would have been seen. Superficial structures were lost from cells which were parasitized (Figure 11) but not from adjacent cells that were uninfected (Figure 12). The loss Of these structures was probably caused by changes in the host cell rather than by direct effect of the organism on these structures. If the host cells were not parasitized, these structures remained intact. Many investigators report that a characteristic Of mycoplasmas is pleomorphism as well as variation in size (Chu and Horne, 1967; Dmochowski, 1967; Freundt, 1960; Hayflick, 1968; Hummler and Armstrong, 1967; Reagan at aZ., 1967; White et al., 1954). Most Of these workers studied organisms that were grown in broth, chick embryos, or tissue culture, except for those in human tissues. The organisms Observed in this study were consistent in shape and structure. The only variation was in size, but this may have been caused by crowding, a difference in the age of the organism in its growth phase, or by sectioning. There were no file- mentous forms or other unusual shapes. 54 Histochemistry In the study of Mycoplasma gallisepticum infection of the turkey sinus epithelial cells, the assumption must be made that each of the animals on the experiment will respond to the microorganism in a similar fashion. Variation in the response between the 2 biological systems was mdnimized by selecting host animals from inbred lines and using a myc0p1asma strain of proven genetic stability in the animal. The absence Of any known unique function of these epithelial cells suggests biochemical activity Of no more significance than maintaining the integrity of the membrane. Under normal conditions little more energy is required by these cells than that which can be derived from the tricarboxylic acid cycle (TCA). Succinic DH, ICDH and MDH, which were found to be active in these cells, are members of the TCA cycle which is localized in the mitochondria (Green, 1960). Mitochondria are found in most cells, and the TCA cycle enzymes function to aid cells in meeting their basal energy requirements. Moreover, the presence of ICDH indicates that these cells are using this cycle as their energy source (Henderson, 1965). The absence Of G6PDH, the first reaction in the pentose shunt, sug- gests that the cells are not utilizing this pathway to Obtain energy. Lactic dehydrogenase, the fourth enzyme, is active in the reversible reaction of pyruvic acid and lactic acid (Lehninger and Wadkins, 1962). Henderson (1965) suggests that the activity of this latter enzyme may regulate the ratio between oxidized and reduced diphosphOpyridine nucleotide. The appearance of succinic DH and MDH granules outside the cell suggests that the integrity of the cell membrane was interrupted by the infection, allowing the mitochondria to pass out of the cell. These 2 55 enzymes are not soluble and have never been prepared mitochondria-free. Pearse (1960) found that succinic acid and malic acid dehydrogenases were the most reliable from the histochemical point of view for demonstration, of TCA cycle activity. The appearance of AP at this stage Of infection probably indicates the invasion of macrOphages which carry this enzyme in the lysosomes (Hirsch, 1961). The localized AP activity in the capil- lary walls probably indicated the location of migrating heterophils. The absence of acid P indicates that necrosis and cellular degenera- tion were absent or only minimal at this stage of the infection but acid P could be expected to appear under more severe conditions (Weber, 1961; Fennell, 1966). Since no measurable acid P was found, this is consistent with Jungherr's report (Jungherr, 1949) and the electron microsCOpic Observations that necrosis is minimal in mchplasma sinusitis. The consistency in enzyme activity of control cells at 5 weeks Of age and at 10 weeks indicates that these enzymes are necessary for intra- cellular function regardless Of the age of the host and do not increase in number or disappear with changes-in age. The intensities of enzyme reactions were unaltered despite the possible increase in energy demands that might be expected because of infection. Mitosis, which must occur when cellular hyperplasia is found, apparently did not affect the enzyme activity. SUMMARY The epithelial lining cells of infraorbital sinuses from turkey poults infected with Mycoplasma gallisepticum (8-6) were stained to F demonstrate the activity of selected enzymes from the hydrolytic, pro- teolytic, and oxidative groups. Only enzymes characteristic Of basic ) . .25“;— energy-generating pathways were found-—succinic acid, isocitric acid, fl lactic acid, and malic acid dehydrogenase. Enzyme activity did not F1 increase or decrease with infection, epithelial cell hyperplasia, or increase in host age from 5 to 10 weeks. The myCOplasma-infected cells were also examined with an electron microscope. The organisms were found in large numbers during the first Week of infection but were fewer in number by the sixth week. The infection was uncomplicated by secondary invaders during the first week, but by the sixth week bacteria and fungal spores had appeared. Myco- Plasmas were lying between cells and within cells, but relatively few were free in the exudate. Cellular degeneration was limited to the distal Part Of the cell. The nuclei and bases of the cells were not affected. Such cells would probably recover if the infection were removed. 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