Mycoplasma-virus interactions in mouse tracheal organ culture

The growth characteristics of Mycoplasma pulmonis and influenza A/PR-8 virus studies in a mouse tracheal organ culture system. M. pulmoins grew well in the tracheal organ culture producing inhibition of ciliary activity. The organism grew in close association with the epithelial cell membrane producing cytopathology as revealed by studies employing stained tissue sections, fluorescent antibody, and electron microscopy. The M. pulmoins did not grow in the culture fluid medium alone but grew when viable tracheal explants were present. However, cultures of heat treated explants with added cholesterol or horse serum supported growth of the mycoplasma. The M. pulmoins was shown to produce hydrogen peroxide. The addition of catalase to the mouse tracheal organ cultures infected with this organism appeared to protect the explants from the toxic effects produced by mycoplasma as indicated by inhibition of ciliary inactivation. This might indicate that the hydrogen peroxide production of the mycoplasma plays a role in the pathogenesis of this organism for the tracheal tissue. There was no ciliary inhibition in cultures that were maintained in medium in which M. pulmoins had grown but was removed by filtration and treatment with antibiotics. This indicated that the organisms must be present to produce ciliary inhibition and that stable toxic products produced by the organism were probably not responsible for this effect. Influenza A/PR-8 virus was also shown to grow and produce cytopathology in mouse tracheal organ cultures. Electron microscopy showed that the virions attached to and caused clumping of the cilia. The virus infection eventually resulted in complete desquamation of the epithelial surface of the tracheal tissue as revealed by stained histological sections. The addition of receptor destroying enzyme (RDE) to the medium was found to be significantly inhibit the growth of the virus in this system. When tracheal organ cultures were dually infected with both organisms, ciliary inactivation and tissue damage occurred earlier that when the organisms were cultured separately. The present of the virus appeared to have no effect on the growth of the mycoplasma, however the mycoplasma inhibited virus replication. The M. pulmoins may have inhibited the influenza virus growth by affecting the nucleic acid or amino acid metabolism of the tracheal epithelial cells. The mycoplasma could also have induced or caused a priming effect on an interferon response. The interferon could then have inhibited the viral replication. Data from this study suggests the possibility of mycoplasma-virus interactions occurring in respiratory infections of man.

MYCOPLASMA-VIRUS INTERACTIONS IN MOUSE ~RACHEAL ORGAN OULTURE by stephen Carl Westerberg A dissertation submitted to the faculty of the Unlverstiy of Utah in partial fulf1llment of the requirements for the degree of . Doctor of Philosophy Department of Miorobiology Un1vers1ty of Utah A.ugust, lQ7l This Dissertation for the Doctor of Philosophy Degree by Stephen Carl Westerberg has been approved July, 1971 o 2� . U0� (� Ohairman, . Supervisory Oo m:mrttee Supervl'sory Oommittee Supervisory Committee �.AA�ry f4h�m s Oomm 1tee � ... . Head, -:., t? � ?' Major Department •• : The author would like to express his appreoiation to Dr. Bill B. Wiley and Dr. Oharles B. Smlth for their . guidance throughout the course of these invest1gat1ons and the preparat10n of this thes1s. The helpful sugges- t10ns of Dr. Louis P. Gebhardt, Dr, Douglas W. Hill and Dr. Barry O. Oole are also appreoiated. The author would also l1ke to express his grat1tude and appreoiation for the cont1nual support and enoourage- ment extended to him by his w1fe, Susan. The help of Dr. OynthlaJensen and Virgin1a Viers 1n the preparation and interpretation ot the eleotron miorographio studies and the teohnioal assistanoe of Fay Fehr in preparation of the histological seot1ons is gratefully aoknowledged. The author wishes to thank Aloma Kern for typing and proofread1ng th1s manusor1pt. This work was supported 1n part by a grant trom the Public Health Service No. HE AL 13915-05. lil TABLE OF CONTENTS ABSTRACT • • • • • • • • • • • • • • • • • • • • • INTRODUCTION • • • • • • • • • • • • • • • • • • • REVIEW OF LITERATURE • • • • • • • • • • • e • • • I. MYcoplasma Pulmonis Infection in Mice • II. III. IV. I. II. III. IV. V. VI. VII. VIII. IX. x. XI. RESULTS I. • 1 • 3 • 3 ., • •• 8 • • • • 14 • • • • • • • • • 21 • • • • • • • • • • • • • • • 29 Influenza Yl!J!! Infections of ~11ce Mycoplasma-Virus Mixed In);ect1ons Tracheal Organ Cultures MATERIALS AND METHODS preparation of Organ Cultures •••••• 29 • • • • • • • • • • • • • • • 30 • • • • • • • • • • • • • • • • • • 30 Mycoplasma Virus • culture Medium • • • • • • • • • • • • • Mycoplasma Quantitation Virus Quant1tat1on • • • • • • • • • 31 • • • • • • • • • • • 32 Inoculation of Organ Cultures Histology 31 • • • • • • 33 • • • • • • • • • • • • • • •.• 33 Demonstration of Peroxide production Electron Microscopy • • 34 • • • • • • • • • •• 35 Fluorescent-Antibody Studies • • • • • • 35 •••••••••••••••••••••• 38 MYcoplasma Pulmonis in MOuse Tracheal Organ CuI ture ,• • • • • • • • • • • • •• 38 • • • • • • • • 38 • • • • • • • • • • • 38 A. Growth of M. Pulmonis B. Histopathology C. Immunofluorescence iv • • • • • • • • • 41 ~ ·. Electron Microscopy • • • • • • • • Effect of Receptor Destroying Enzyme • Effect of Viable and Heat Treated Tissue on Growth of 11. Pulmon1s • • • • D. , E. F. Effect of Medium Supplements G. H. II. Effect of Catalase 57 • • • • • • • • • • Influenza A Virus in Mouse Tracheal Organ Culture • • • • • • • • • • • • • • • A. Growth of Influenza A CPR-8) • • • • • 55 62 62 Histopathology C. Immunofluorescence D. Electron Microscopy E. Effect of Receptor Destroying Enzyme • 70 F. Combined Infection with ~. Pulmonis and Influenza A. • • • • • • • • • • • 73 G. Histopathology •• • • • • • • • • •• 82 H. Electron Microscopy • • • • • • • • • • 87 • • • • • • • • • • • • • • • • • • • • • 91 SUMMARY • • • •••••••••••• 52 B. DISCUSSION 64 • • • • • • • • • • 64 • • • • • • • • • 66 • • • • • • • • • • • • • • • • • • • • 112 • • • • • • • • • • • • • • • • • • 114 • • • • • • • • • • • • • • • • • • • • • • • • 125 LITERATURE CITED VITA 46 49 0 J. 45 • • • • • Infected Culture Filtrate Effect on Ciliary Activity • • • • • • • • • Demonstration of Hydrogen Peroxide Product:ton • • • • • • • • • • • • • · .. I. 42 v LIST OF TABLES TABLES I. II. III. IV. V. VI. VII. VIII. IX. x. XI. MYCoplasma eulmon1s Infection of Mouse Tracheal Explants in Culture • • • • • • • • • Effect of Receptor Destroylnq Enzyme (RDE) on MYcoplasma pulmonis Infection of Mouse Tracheal Explants • • • • • • • • • • • • • • MYcoplasma pulmon1s in Organ Cultures of Viable and Heat Treatea t~use Tracheal Explants • • • • • • • • • • • • • • • • • • • Effect of Madium Supplements on the Yield of MYcoelasma pulmon1s from the supernatant Medium of Heat Treated Tracheal Explants • •• Effect of sterlle Culture Filtrates from ~. lUlmonis Infected Cultures on' Ciliary Act vity of Un1nfected Mouse Tracheal Explants • • • • • • • • e • • • • • • • • • • Effect of Added Catalase, Glucose, and Am1no-Triazole on~aoplasma pulmonis Infection of Mouse Trae eal Organ Cultures • • • • Influenza A (PR-8) Virus in Mouse Tracheal organ Culture • • • • ' • • • • • • • • Effect of Receptor Destroying Enzyme (RDE) on Infection by Influenza A in Mouse Tracheal Organ CUlture • • • • • • • • • • • • • • • • ,Mlccplasma eulmonis Followed by Influenza A Virus in Mouse Tracheal Organ Culture • • • • Influenza A Virus Followea ~. MYcoplasma Rulmonia in Mouse Tracheal Organ Culture • • • Influenza A Virus and trt:eoElasma eulmonis in Mouse Tracheal Organ Culture • • • • • • • vi 39 47 50 53 56 60 63 71 74 79 83 LIST OF FIGURES FIGURES 1. 2, Appearance of Mouse Tracheal Explants After 5 Days Incubation. • • • • • • • • • • • Electron Microgra·ph of Normal Mouse Tracheal Explant After 1 Day in Culture • • • • • • •• -PAGE 40 43 Mouse Tracheal EXplant After Electron Microqraph of tl. eulmon:La Infected 7 Days 1n Culture 44 Yield of ~. ~~m?~ from Supernatant Medium of Mouse Tracheal Organ Cultures Treated With Receptor Destroying Enzyme (RDE) • • • • • •• 48 Yield of M. pulmonis from the Supernatant Medium of Cultures Containing Viable and Heat Treated Mouse Tracheal IXplants • • • • • • e 51 Effect of Medium supplements on the Yield of ~. Rulmonis from Supernatant Medium of HOAt Treated Mouse Tracheal Explants e • • •• 54 Methylene Blue sta1ninq of'Erythrocytes surroun~ing a Colony of M. pulmonis Indicating Hydrogen Peroxide Production • • • • • •• 58 Effect of Added Catalase, Glucose, and Am1noTriazole on the Ciliary Activity in M. pulmon1.s Infectea Tracheal Orqan CUltures •• 61 Appearance of Mouse Tracheal Explants After 5 Days Incubation • • • • • • • • • • • • •• 65 10. Mouse Tracheal Explant Infected In Culture With Influenza A Virus After 5 Days Incubation 67 11. Electron Microqraph of Influenza A/PR-S Infected Mouse Tracheal Explant After 3 Days In Culture • • • • • • • • • • • • • • • • •• 68 3. 4. 5. 6. 7. s. 9. 12. 13. Electron Micrograph of a Cluster of Influenza A.!PR-S Virus • • • • • • • • • • • • • • Effect.of Receptor Destroying-Enzyme (RPE) on the Yield of Influenza A Virus frOm the supernatant Medium of MOuse Tracheal Or9an Cultures • • • • • • • • • • • • • • • • • • • vii 69 72 FIGURES 14. 15. 16. 17. 18. 19. 20. 21. 22. Yield of ~. pulmon1s and Influenza A Virus from supernatant Mediwn of Dually Infected Mouse Tracheal Organ Cultures When~. eulmon1s Was Inoculated F.1rst • • • • • • '. • • percentage of.'lracheal Explants Show1nq Absence of Ciliary Activity When Infected with M. pulmon1s Followed by lnfluenza A • e . - e • • • 77 Yield of 1:1. pulmon1s and Influenza A Virus from supernatant Mea1um of Dually Infected Mouse Tracheal Or9~n Cultu~es When Influenza A Virus Was Inocul.ated First ... e • • • .. . . . eo percentage of Tracheal Explants Showing Absence of Ciliary Ace1v1ty When Infected W1~h Influenza ~PR-9 Followed by ~. eulmonis • • • 81 Yield of ~. pulmon1s and Influenza A Virus from Supernatant 14edlum of. Dually Infected MOuse Tracheal organ Cultures When Both Orqanisms are Inoculated at the Same Time. • • • •• 84 76 Percentage of Tracheal Explant~ Showing Absence of Ciliary Activity ~ihen Infected With Influenza A/PR~ an4 ~. 2ulmonis at the Same Time • • • • • • • • • e e e • • • • • Appearance of Mou.se Tracheal Explant After 5 Days In Culture • • • • • • • • • • • • • •• 86 Appearance of Mouse Tracheal Explant After 5 Days In Culture • • • • • • • e • • • • • •• 88 Electron Microqraph of ~. ealmon1s and Influenza ~PR-a Virus Dual Infection of Mouse Tracheal Explant After 1 Day in culture •.••• ,a9 viii 8S ABSTRACT The growth characteristics of Mycoplasma pulmonis and influenza' A/PR-8 virus were studied in a mouse tracheal organ culture system. M. pulmonis grew well in the tracheal organ culture producing inhibition of ciliary activity. The organism grew in close association with the epithelial cell membrane producj.ng cytopathology as revealed by studies employing stained tissue sections, fluorescent antibody, and electron microscopy. The M. pulmonis did not grow in the culture fluid medium alone but grew when viable tracheal explants were present. However, cUl- tures of heat treated explants with added cholesterol or horse serum supported growth of the mycoplasma. pulmonis was shown to produce hydrogen peroxide. The~. The addition of catalase to the mouse tracheal organ cultures infected with this organism appeared to 'protect the expla~ts from the toxic effects produced by mycoplasma as indicated by inhibition of ciliary inactivation. that the hydroq~n This might indicate peroxide production by the mycoplasma plays a role in the pathogeneSiS of this organism for the' tracheal tissue. There was no ciliary inhibition in cultures ,that were maintained in medium in which M. eulmonis had been grown but was removed by filtration and treatment with antibiotics. This indicated that the organisms must be present to produce ciliary inhibition and that stable toxic products produced by the organism were probably not responsible for this, effect. Influenza A/PR-8 virus was also shown to grow and produce cytopathology in mouse tracheal organ cultures. Electron microscopy showed that the virions attached to and caused clumping of the cilia. The virus infection eventually resulted in complete desquamation of the epithelial surface of the tracheal tissue as revealed by stained histological sections. The addition of receptor destroying enzyme (RDE) to the medium was found to significantly inhibit the growth of the virus in this system. When tracheal organ cultures were dually infected with both organisms, ciliary inactivation and tissue damage occurred earlier than when the organisms were cultured separately. The presence of the virus appeared to have no effect on the growth of the mycoplasma, however the mycoplasma inhib1ted virus replication. The~. pulmonis may have inhibited the influenza virus growth by affecting the nucleic acid or amino acid metabolism of the tracheal epithelial cells. The mycoplasma could also have induced or caused a priming effect on an interferon response. The 1nterferon could then have inhibited the viral replication. Data from this study suggests the possibility of x mycoplasma-virus interactions occurring in respiratory infections of man. xi INTRODUCTION Natural resistance to respiratory infection is associated with the capacity of the respiratory tract to protect itself against bacteria and viruses. specific host defenses are: (1) Some non- surface fluids such as mucus, surfactants and lysozyme, (2) mechanical removal of infecting organisms by the pulmonary phagocytic activity of alveolar macrophages and the highly specialized mucociliary apparatus (Green, 1968). It has been suggested that ciliostasis in the respiratory tract is of great significance in the development of pulmonary infections (sang, 1961). Although much work has been reported concerning the growth of respiratory mycoplasmas and viruses in cells cultured in monolayers, the growth characteristics of these organisms in differentiated tissues of the respiratory tract are not well defined. Employment of a tracheal organ eulture system to study the role of infection in respiratory disease seems alog1cal extension of previous studies in cell cultures. Organ cultures offer an opportunity to study the effect of infectious agents on the ciliary activity, one of the natural host defenses found in the whole animal. They also permit experimental conditions to be varied much more than the homeostatic reactions of an intact organism. It is also possible to study cellular 2 changes induced organ cultures. by~infectious agents much more easily in Infections'in tracheal organ cultures may have characteristics which are unlike those seen in infections studied in tissue culture but nevertheless bearing on the mechanisms ,operating in ~. The purpose of the research to be described was to determine the effects produced by a respiratory mycoplasma (MYcoplasma pulmonis) and a respiratory virus (influenza A strain PR-8) in a mouse tracheal organ culture system. Studies are also described relating to possible mechanisms of pathogenesis of H. pulmonis in organ culture. Finally a mixed infection by both organisms in the organ culture will be described and a possible role of synergism in mixed infection discussed. REVIEW OF LITERATURE I. MYCOPLASMA PULMONIS INFECTION IN MICE In order to understand the action of ~. pulmonis in tracheal organ culture. some of the characteristics of this mycoplasma should be described. MYcoplasma pulmonis, originally designated L3 after the Lister Institute, is a pleomorphic organism of 0.5 to 1.0 micron in diameter (Tully, 1965). The pleomorphic nature is possibly the result of the absence of a cell wall. This organism is easily isolated from infected rats and mice in which it frequently causes a chronic pulmonary disease. Tully (1965) published a detailed description of the properties of murine mycoplasmas. reported for ~. Some of the properties he pulmonis were as follows: (1) ~. pulmonis grows on agar media equally well aerobically or anerobically in 2-4 days producing granular colonies lacking a well defined center, (2) it will reduce methylene blue, ferment glucose, maltose and dextrin, and (3) it produces beta hemolysis of sheep or guinea pig red blood cells. and Tully (1970) established that for growth of cholesterol is absolutely required. ~. Razin pulmon1s They found that the organism would grow in serum-free medium if the medium was supplemented with albumin, L-arginine, palmitic acid and 4 cholesterol. Nelson (1960) reported that well in HeLa cell C\,J,tt''''es and c~nlc1 H. pulmonis grew be seen microscopi- cally in the fluid phase and on and within the HeLa cells. A description of. diseases in mice caused by ,M. pulmonis may help in understanding its action in organ culture and possible role in a cyccplasma-vir.t'" dual infections. Nelson (1937) was first to describe the disease infectious catarrh of mice. He isolated what he called "cocco'bacilliform bodies" from a nasal exudate of infected Swiss mice. Nelson (1948) later described a middle ear infection caused bf the mycoplasma. He felt that this was the most common manifestation of the mycoplasma disease produced under experimental conditions in mice. Nelson (1958) more recently called this disease the most prevalent PPLO (pleuropneumonia-like organism) disease of mice. He reported that the disease is characterized by a persistent exudative inflammation of the nasal 'passages, middle ears and lungs. There was a copious, mucopurulent exudate pro- duced in the nasal passage but was rarely discharged from the anterior nares. He also observed that the resulting exudate accumulated in the tympanic cavity and remained there throughout the life of the host. A state of equilib- rium between the host and the infecting mycoplasma was reached which endured for months. When the mice finally died from the infection, many of them showed no sign of pulmonary involvement, however, most of the mice showed 5 siqns of mucopurulent inflammation of the bronchial mucosa sometimes plugging the lumen. Cotchin and Roe (1967) described a disease of mice called qrey lung disease which was caused by mycoplasma. The infected lungs showed partial or complete red-grey consolidation of one or more lobes. Cut sections of the lungs revealed mononuclear cuffing, edema, swelling and proliferation of cells in alveolar walls and in the alveoli. Nelson (1962) concluded that chronic respiratory disease (CRD) in laboratory mice was a cyndrome resulting from two separate diseases, infectious catarrh caused by ~e pulmon1s and enzootic bronchiectasis caused by a virus. The distinguishing features of the disease were pneumonia plus purulent inflammation of the nasal passages, the middle ear and the inner ear. long duration. The disease was of slow onset and Nelson also stated that recovery from eRn had never been observed, however, deaths due to the disease were usually sporadic and limited to older animals. Lutsky and Orqanick (1966) studied mycoplasma pneumonia in gnotobiotic mice. They found that conventional and qnotobiotic mice inoculated intranasally with ~. pulmon1s had similar peak mortalities between the 5th and 12th day. The microscopic pathology of the lung lesions for both groups were also the same. When they inoculated the conven- tional mice with sterile saline they isolated mycoplasma' from their lungs. However inoculation of sterile saline into 6 gnotobiotic mice did not result in mycoplasma isolation. In another study, Lutsky and Organick (1968) demonstrated the high degree of host specificity of certain mycoplasmas. pulmonis, H. They inoculated three groups of mice with pneumoniae, and weeks they isolated ~. ~~ H. salivarium. of 3S mice. After four pulmonis from 65 out of 79 mice, pneumoniae from 3 out of 34 mice and o out H. ~. sa11var1um from Their data suggested to them a high degree of host specificity of the mouse specie, ~. pulmonls, since mice were the only hosts that they tried to infect with the H. pulmoniS. It would be difficult to conclude that this study demonstrated a high degree of host specificity of the mycoplasma. Gilbey and pollard (1967) attempted to isolate mycoplasmas from germ-free mice but were unsuccessful. M,ycoplasmas could not be found in specimens of thymus, lung, liver, spleen or bone marrow removed from 47 leukemic germfree SW1ss-Webster mice. Seroloq1cal tests also failed to indicate that the mice had been exposed to mycoplasma infection. They did not say which mycoplasma specif1c anti- bodies were looked for. H. pu1monis has also been reported to cause polyarth- ritis in mice by Barden and Tully (1969). challenge with!!. pulmonis (JB Intravenous s.train) produced joint in- volvement in all the mice 1noculated and the organism was recovered frequently from the enlarged Joints. The 7 polyarthritis resolved slowly but some residual jOint swelling was noted for as long as four months. Mrcoplasma-virus mdxed infections of mice have also been reported. When Horsfall and Hahn (1939) infected mice with an unidentified murine pneumonia virus they could consistently isolate mfcoplasma from suspensions of the consolidated mouse lungs. They found, however, that mix- tures of the mycoplasma plus the virus did not increaso the extent or severity of the pneumonia over that produced by the virus alone. Brennan et ale (1969) suggested that typical murine pneumonia was caused ~ Pasteurella pneumotropica and ~ a mixed infection pulmonis. ~. their data indicated that the effect produced Analysis of ~ the two organisms was an additive effect rather than a synergistic effect. Fluorescent antibody studies revealed that the Rulmonis localized in the bronchial epithelium and the ~. t. pneumotropica localized in the alveolar lesions. It appears then that ~. pulmonis causes infections in mice and the most common infection caused was located in the respiratory tract. The respiratory infections were usually subclinical unless induced as saline. A secondary infection ~ ~ stress factors such a respiratory virus might cause a more severe disease if it also induced a concurrent H. pulmonis infection. 8 II. INFLUENZA VIRUS INFECTIONS OF MICE A knowledge of a few general characteristics of the influenza virus will be useful in interpreting the ac~ion of this virus in a mixed infection in mouse tracheal organ culture. Influenza virus belongs to the myxovirus group of viruses. The myxoviruses contain a single stranded ribo- nucleic acid (RNA) core surrounded by a protein capsid enclosed in a lipid envelope. The envelope has morpholo- gical structures called spikes which aid in the attachment of the virus to host cells and neuraminidase enzyme embedded in its surface which functions in release of the virus from the infected cell (Marcus and Schwartz, 1968). A description of cell receptor sites for the influenza virus attachment may be helpful in understanding mycoplasmavirus interactions. Mycoplasma possibly utilize the sarna receptors for attachment to the host cells. Fazekas de st. Groth (1950) was first to demonstrate that the neuraminidase was responsible for release and not attachment of the virus as previously thought. Marcus et ale (1965) demonstrated that the surface of cytoplasm-free nuclei also contained specific receptors for the attachment of the myxovirus. Marcus and Schwartz (1968) proved that the N-acetylneu~am1nic acid attachment sites on the plasma membrane were not essential for normal cell function since cells grew normally in concentrations of neuraminidase sufficient to maintain 9 destruction of the receptors. l~ny species of mycoplasma can metabolize arginine pro- ducing ammonia (Schimke and sarile, 1963). The production of ammonia could adversly affect the replication of influ- enza virus. The presence of glutamine and ammonia inhibit the replication of influenza virus reported Eaton and Scala (1961). They felt that the was at the 1nhib'.~~ryaction early stage of viral replication and did not affect virus attachment to susceptible cellse Yamashiroya et ale (1965) also reported that ammonium ions in concentrations of 57 and 63 micrograms per ml present in co~~ercial lots of medium #199, inhibited the cytopathic effects of influenza B virus. They speculated that the high levels of ammOnium ions could have resulted from the decomposition of glutamine during storage of the growth medium. The influenza A mouse adapted strain. PR-8 employed in this study was a The mouse adapted strains of influen- za viruses differ from the wild-type virus e Influenza A viruses can be easily adapted to growth in mice. Borecky et ale (1966) after studying the process of adaptation concluded that the process of adaptation represented a selection of virions with more suitable properties for their replication in a new host. In the new host enVironment, mutants were formed spontaneously which had replaced the original population. Chu (1951) found that mouse adapted strains were resistant to neutralization ~ normal mouse 10 serum whereas nonadapted strains were not. He stated that the increased resistance of influenza virus to neutralization by normal mouse serum accompanied the process of mouse adaptation. A review of the observations reported concerning the pathogenic process of the influenza virus infection of mice may help interpreting the action of the virus in dual infections in trachea], organ culture. Nakamura (1965) stUdied the pathogenicity of influenza A and B strains in mice and found that 6 out of 13 strains were not pathogenic, 3 strains were pathogenic only when inoculated in large doses and the remaining 4 strains were pathogenic in small doses. Dolowy and Muldoon (1964) studied influenza infection in germ-free mice and found that these mice were more suscept1blethan conventional mice to influenza virus infection as indicated by more severe clinical illness and increased mortality. However, the mortality difference they reported was small. Taylor (1940) reported on the experimental infection of mice with influenza A PR-8. He found that when mice were inoculated intranasally the maximum titer of the virus in the lungs was reached within 24 hours. The maximum titer was always reached before the appearance of gross lesions in the lungs. The injury inflicted by the virus was con- fined to the infected cell, he reported, and death of the host occurred only after a sufficient proportion of the cells of a vital organ had been affected preventing proper 11 functioning of that organ. Hers et ale (1962) described histological and fluorescent antibody studies of mouse influenza A virus infections and reported within 4-6 hours after intranasal inocculation, virus antigen was detected in the bronchial epithelial cells. changes were seen at 2~ hours an~, The first morphological conSisted of necrosis of bronchial epithelial cells and interstitial inflammation of the lung. Macrophages appeared after 24 hour,s but were not numerous after 48 to 72 hours. The alveolar cell l1ning showed progressive viral cytopathic changes comparable with the changes seen in the bronchial ciliated cells. They concluded that influenza virus pneumonia in mice can begin in the alveoli instead of by spreading down the bronchial tree. Nayak et ale (1964) also studied influenza virus infections of mice and concluded that the initial site of virus infection appeared to be in the bronchial epithelium and infection of the alveoli did not occur until 24 hours postinoculation. The infected cells sloughed, disintegrated, and released virus which in turn infected adjacent celle. They concluded that the virus progressively infected the bronchi, bronchioles, alveolar ducts, and alveoli during the course of infection. Loosli (1949) found well defined consolidation of all 5 lobes of the lungs of mice infected with influenza A PR-8 after 5 days. The single left upper lobe was most frequently involved, he reported. Some mice which showed 30% lung 12 consolidation survived. How the influenza virus produces infections and disease in the host is not completely understood, howeve~, many workers have speculated on possible mechanisms of pathogenesis. Davenport (1961) discussed some possible mechanisms for the pathogenesis influenza viral infections in man. When the vir.us embeds in the mucus covering the respiratory epithelium, the ciliary action generally carries the virus toward the exterior.. However, Gottschalk (1961) has shown that the neuraminidase activity of influenza virus lowered the viscosity of the mucus blanket converting it to a watery fluid thus exposing the cellular surface receptors for virus attachment. This also may pro- mote the spread of virus by the flow of virus-containing fluids to lower portions of the respiratory system, he reported. The highly toxic nature of influenza was discussed by Ginsberg (1951). He reported that the toxic effect was due to the direct action of the virus particle and could be produced by in~ectious as well as ~ inactivated virus. The toxic effect produced cell damage and induced a pyrogenic effect. Fisher and Ginsberg (1956) observed that influenza virus inhibited phagocytOSiS by leucocytes and postulated that this was due to a block in the energy yielding mechanism by inhibition of glycolysis. Regeneration of the epithelium after influenza virus infection started in the early stages of cellular 13 degeneration before (1966). des~uamation o~~urred, observed Hers The regeneruticn in the luter stages of the dis- ease was characterized nonkeratinized ~ stratii~;i,ed appearance of undifferentiated, epithelium. A completely normal ciliated mucus-prOd\1c:i.ng epitheliwn appeared later. Some nonspecific l,,'osistance. Li1ctors which playa role in protecting the mouse from inf),ll.onza virus infections might also play a role in infections in mouse tracheal organ cultures. Some factors which playa role in res:1.s- tance of mice to influenza viral infections are the presence of heat stable mucoprote·ins in respiratory secretions. Davenport (1961) concluded that these ·'alpha inhibitors" were analogous to tissue receptors and thus would combine with virus particles inhibiting their attachment to cells. He reported another inhibitor called beta inhibitor which was a heat labile proteinaceous substamce that was capable of inactivating the viru,s ~ v~,tr,£. Interferon probably plays a major role in recovery the first viral infection. f~om Schulman and Kilbourn (1963) found that the intranasal administration of inactivated influenza virus lead to a state of local resistance which altered the course of a subsequent infection with qous influenza virus. hete~olo­ Their data suqqested to them that the induced interference was mediated ~ interfe~on sinco the action was local in nature and was nonspecific with respect to the viruses i~hibited by its action. This. does 14 not seem to be enough criteria to call the inhibitor interferon. III. MYCOPLASMA-VIRUS MIXED INFECTIONS MUch of the literature dealing with virus-mycoplasma interactions leads one to believe that viruses and mycoplasma grow independently and neither aid nor hinder the growth or replication of the other. explored the possib~lity Armstrong et ale (1965) that mycoplasma species related to MYcoplasma hominis type I might cause resistance of cell cultures to superinfection with vesicular stomatitis virus (YSV). Cell cultures infected ~lith the mycoplasma sho\"Tecl delayed cytopathic effect (CPE) on challenge with VSV. This dealy was found to be caused by lowering of the pH by the mycoplasma. No resistance to veSicular stomatitis virus (YSV) was evident when the infected cultures were maintained at the same pH as the controls. Sakamoto (1969) reported that ~. Nakamura and hominis type I had no effect on the growth of influenza virus. They reported that replication of influenza virus was not inhibited when the virus was inoculated before or at the same time as the mycoplasma. Schulze and Schlesinger (1963) found that suspensions of dengue type 2 virus were of equal titer when assayed employing mycoplasma contaminated and mycoplasma free KB cells. stock and Gentry (1968) observed that Mycoplasma\hominis contributed significant deoxyribonuclease 15 activity to otherwise normal or virus-infected cell cultu~eB. They found, however, that this activity did not destroy the replicating viral genome of equine herpes virus (EHV) therefore the virus repl:l.cated successfully despite the presence of the contaminating mycoplasma. Kagan (1967) also reported a situation in which mycoplasma apparently had no effect on virus gro~~he He isolated m¥coplasma species contaminating HeLa and A-l cells and infected chick embryo fibroblast cell cultures with these organisms. After periods of 1, 2, and 3 days the infected cell cultures were challenged with Eastern encephalomyelitis virus (EEE), venezuelanencephalomye11tis virus (VEE) and Newcastle disease virus (NDV). He found no viral growth inhibiting effect in the contaminated cultures compared with mycoplasma. free control cultures. Mohamed et ale (1969) studied a mixed mycoplasma-virus infection in turkeys. toqether with influenza When 11. meleaqr1dis was inoculated A virus there was no synergistic effect (i.e., mixed infection produced more severe disease) as revealed by clinical signs, incubation periods or necropsy findings. They pointed out that this did not preclude a synergistic effect under a different set of conditions. Singer et ale (1969) found that a greater yield of VSV virus was obtained from chick embryo fibroblasts cells infected with MYcoplasma arqinini than from uninfected cells. 16 They reported also, that similar results were obtained with ~. qyorhinis contaminated cell cultures. Heishman et alo (1969) demonstrated a virus-mycoplasma mixed infection !a !!!2. They found chickens infected with Newcastle disease virus (NDV) or infectious bronchitis (IS) virus together with 1:1. gallisepticum. bad a higher- mortality rate than bir.d.s infected with eithei~ of the agents alone. The mor.tality resulting from airsacculitis was increased five-fold ovor the controls when !:1" qallisept1cWl! was introduced tlith the virus. In a simila~ report, Ranck et ale (1969) found that a combination of ~. ga~liseRtic~ and avian influenza A strain meleagrium injected into the thoracic air sacs of turkeys caused more extensive lesions in the air sacs than were produced by either agent alone. However, no syne~91s­ tic effect was noted when 1:1. gallisepticum "'tas injected with avian paramyxovirus (strain yucaipa) into turkeys under Similar conditions. Milligan and Fletcher (1969) studied the effect of ~. pneumoniae on rhinovirus RNA synthesis in KB cells. Viral RNA synthesis was stimulated throughout the entire period of observation in cells previously infected with ~. pneumoniae. Their data indicated to them that the enhancement was not produced by an extra-cellular element but required the presence of intact mycoplasmas. The initiation of stimulation appeared to occur at an early stage in the viral repli~a.tion cycle. They felt that their 17 observations might indicate that the interaction of ~. pneumoniae with rhinovirus could result in a more severe upper respiratory tract infection. There are also reports concerning viral onism caused by mycoplasmas. 9r~~h antag- The work of Brownstein and Graham (1961) indicated that even though the growth of L cells in suspension ~coplasma, 'i'~as unaffected 11Y the presence of the yields from single-burst experiments of Mengo virus were very low as compared with virus yields from mycoplasma-free cells. They also pOinted out that the mycoplasma infected cells, when infected with virus, seemed unduly fragile which could have accounted for the periodic decrease in plaque titer of the stock virus. Rouse et ale (1963) reported that mycoplasma which were able to metabolize arginine apparently 1nterferred with the viral infectious process of adenovirus type 2 resulting in low plating efficiency by depriving the cellvirus complex of a single essential nut~ient arginine. They also observed that adenovirus types 1, 3 and 4 wore also inhibited ~ mycoplasma. was affected by the presence of and Leach (1963) reported. with this ~coplasma The growth of measles virus ~. ga1l1sept1cum, Butler They infected HEp-2 cultures then superinfected the cultures with measles virus two days later. After 9 days the virus titer in the infected cells was only 104 • 5 TCIDSO compared with 105 • 5 in mycoplasma free cells. At 14 days the cells 18 infected with mycoplasma plus the virus were completely degenerated. Mycopl~~",,~-free showed no CPE. vir.'(.'U1 infected cul tU1:'es They also found that measles virus 9ro~~h was not affected by the presence of the laboratory strain of ~. hominis type I. Somerson and Cook (1965) reported that the multiplicat.:ton of Rous sar.coma virus (RSV) and Rous associated of ~. vir\~r. (RAV) was inh:thited by the presence orale I in WI-26, WI-38 and chick-embryo fibroblast cell cultures. In everyone of their experiments titer reductions of at least 1 to 3 logs were observed in RSV assays in mycoplasma infected cells. suppression of RAV ~ They demonstrated their failure to detect avian leukosis CF antigen in mycoplasma infected cells inoculated with RSV. Inhibition of RSV did not appear to be a non- specific suppression of replication, they concluded, since cell~ infected with the mycoplasma supported the multipli- cation of influenza B, Taiwan strain. In contrast, Nakamura and Sakamoto (1969) reported that the growth of human influenza A, strain WS, was greatly inhibited ~ mixed infection with H. pneumoniae when the virus was inoculated two days after the mycoplasma inoculation. However, they noted only a slight reduction in viral replication when pneumoniae. ~. orale was substituted for ~. They felt that this effect might be due to depletion of arginine in the medium. The work published by Afshar (1967) revealed that cells previously infected with 19 ~. boviqen1talium would not support the same degree of growth of infectious bovine rhinotrache1tis (IBR) virus as mycoThe growth rate of the IBR virus was plasma-free cells. initially delayed although the final titer of the virusproduced did not differ significantly from that produced in mycoplasma-free cultures. hours in the appear('''''~e of They no~ed cytopn~:')j.c a delay of about 25 effect produced by the virus in cultures of calf kidney cells infected with mycoplasma. Fogh (197Q) presented data that indicated that concurrent SV40 and ~. fermentans infection modified the SV40 induced transformation of human amnion (FL) cells. He noted the late appearunce of transformed fOCi, and lesser growth potential of the cell strains resulting from the dual infection. He also noted consistant appearance of new chromosome varieties after exposure of the FL cells to the mycoplasma ~. fermentans indicating to him that the mycoplasma-induced genetic changes were responsible for the reduced transformations. Complete cessation of virus production in mycoplasma infected cell cultures was also noted. A decreased yield of vaccinia virus in cultures pre- viously infected with (1970). ~. arginini was noted by Singer et ale However, no decrease in yield was seen from cul- tures which had been previously infected with Me ax0r.hinis. They believed the decrease in vaccinia yield observed when 20 cultured with H. arginini, reflected the utilization of the arginine in the medium by the mycoplasma e When arginine was added to the dually infected cultures the virus yield was the same as in the controls. Evidence presented by Gafford et ale (1969) demonstrated that the presence of ~~~ galliseet.ieum in cul tux:-es in- fected with fowl pox n:Lr.us (FP) hnQ no effect on the kinetics of the virus growth curve but the was greatly reduced e ~ !!y£ infectivity The pock count of FP virus alone \-Tas approximately 1 log lower than the plaque titer, the pock count of the mixture of mycoplasma plu.s FP was 5 to 6 logs lower. In the dually infected cultures there developed a small-plaque variant of the virus that was not seen in mycoplasma-free cultures. They raised the question that possibly mycoplasma contamination could also be responsible for other instances of altered !B !!Y£ infectivity seen after many passages of viruses in tissue culture. Ragan (1967) studied the effect of mycoplasma on the growth of some RNA viruses in chick embryo fibroblast cells. He found that the growth of Venezuelan encephalomyelitis virus (VEE) and Newcastle disease virus (NOV) was greatly inhibited and H. ~ the presence of hominis type 1. ~. ~. laidlawi1, M. egalact1ae sa11var1um inhibited the viruses only if the mycoplasma inoculation preceeded the virus inoculation b¥ 3 days. The only reports found of the effect on mycoplasma 21 growth produced by virus infected cells in vitro was that of Nakamura and Sakamoto '(1969). They noted that Japanese encephalitis (JE) or human influenza A virus when inoculated into porcine kidney cell cultures infected with or ~. ~. pneumoniae orale had a stimulating effect on the growth of the mycoplasma. Nelson (1958) reported that the normal mouse brain does not provide optimal conditions for the growth of most mycoplasmas. However, if mouse hepatitis virus is injected simultaneously with the mycoplasma, the growth of the mycoplasma is markedly increased. There was no indication of specificiety in relation to the type of mycoplasma used. He pointed out that multiplication of the virus in the brain was an absolute prerequisite for the mycoplasma growth stimulation. These studies indicate that mycoplasma often do interact with viruses in cell cultures. A few reports even indicated that dual infections in animals have been produced, frequently caus,ing a more severe disease than was produced ~ either agent alone. No studies have yet been found concerning mycoplasma-virus mixed infections in tracheal organ cultures. IV. TRACHEAL ORGAN CULTURES For many years the use of the term tissue culture implied the techniques where small pieces of tissue that 22 were allowed to grow in a medium encouraging the outgrowth of cells. The first successful culturing of tissue outside the body as reported by Hoorn and Tyrrell (1969) was a form of organ culture carried out by Ljunggren (1898) and consisting of human skin maintained for several weeks in ascitic fluid. Thompson (1914) observed that isolated parts of chicken embryos (i.e., toes, feather rudiments and lens) would increase in volume when cultivated in vitro. He called this controlled growth, compared to uncontrolled secondary outgrowth of dedifferentiated cells from the cut surface of tissue. He also distinguished between "unorgani- zed It or cytotypic growth and "organized II or organotypic growth. The term tissue, defined by Hoern and Tyrrell, (1969) means the assemblage of cells the majority of which are of Similar type in an ~rganized arrangement. plex of different types of tissues constitutes an A comorgan~ Tissue culture is used to describe any technique by which cells derived from tissues are processed or propagated ~ vitro. The term organ culture defined by Hoorn and Tyrrell (1969) refers to the methods in which embryonic rudiments or small pieces of adult organs are maintained with ~heir fundamental structure intact. ~ vitro. In a successful organ culture the cells not only retain their appearance, but also function in a specialized way_ of the thyroid gland syn~hesize For example, cells thyroid hormones, cilia on 23 ciliated cells beat, and mucin secreting cells synthesize and release mucin. In organ cultures it is at least cer- tain that the cells studied are those which were removed from the animal and in most cases it can be shown that they have undergone no great morphological change. Organ cul- tures offer an opportunity to study the effect of infectious agents on the differentiated target cells found in the intact host. They also permit experimental conditions to be varied. much more than the homeostatic reactions of an intact organism. It is also possible to study cellular changes induced by infectious agents Which cannot be investigated by biopsy or postmortem. Hoorn and Tyrrell (1969) stated that it appears that virus specificity of organ cultures is more like that of the intact animal than of tissue cultures made from it. Tracheal organ cultures have been prepared from monkey (Barski et al. 1959), human embryo (Hoorn , 1966), swine (aeed, 1969), new born calves (Campbell et ale 1969), rabbits (Hoorn, 1964), chickens (Bang and Niven, 1958), ferrets (Tyrrell and Hoorn, 1965), guinea pigs (Craighead, 1966), hamsters (Collier, 1969), and mice (Willems, 1968). small portions of the trachea containing the Ciliated epithelium have been maintained ~ Nadai (1963) on plasma clots on coverslips in tubes, on rayon nets floating on a liquid medium 1n a watch glass by Bang and Niven (1958), on filter paper or membrane filters on glass beads in petri dishes by 24 Craighead (1966) and on scratched surfaces of plastic petri dishes as described by MCIntosh (1967). Atmospheres commonly used are pure oxygen, air, or in air with 5% C02, air alone being the atmosphere used by most workers. Incubation temperatures usually depend upon the virus or bacterium being studied.: Survival times have been reported to be as little as 5-6 days in the ease of adult human trachea as reported by Maddi (1963) to as long as two months for adult ferret and chick trachea reported by Bang and Niven (1958). The epithelium of the human trachea is made up of pseudostratified columnar epithelial cells with the surface composed of about 5 ciliated cells to each goblet cell (Rhodin, 1966). There are approximately 200 cilia on each 1 cell varying from 5 to 7 microns 1n length and approximately 0.3 microns in diameter (Rhodin, 1966). Cilia beat from 17 to 2S beats per second (Sleigh, 1966). This beating is not synchronous but rather the cilia beat one after another in a metachronous manner with the waves moving at a rate of 100 to 200 microns per second. 'Rylander (1966) reported that mucociliary transport is 'c~pable of clearing the con- ducting airways of inhaled particles in a few hours. Rhodin (1966) reported that ciliated cells are sloughed more frequently than goblet cells, as judged by the number of disintegrating cells. The sloughing of ciliated cells is preceded by a blebbing and swelling of the apical cytoplasm. 25 Subsequently mitrochondria swell and the cytoplasm of the whole cell is thined out. Finally, the base of the cell is retracted and the cell 1s pushed out. Several methods have been reported for measuring ciliary activity. Ballanger et ale (1966) stated that if explants of less than 12 mm diameter are placed on plasma clots and hung upside down in a Rose chamber, then the localized liquefaction of the clot would occur so that the beating cilia propel the aggregate of cells around a constant axis. If undisturbed, the rotation continued for a long time, they observed. A decreased rotation may be caused by failure of frequency of beat, decreased excursion, decreased efficiency of each ciliary cycle, or a loss in metachronal rhythm. Abilit.y of the cilia to perform this work may thus be measured quantitatively. With the use of a stroboscope connected to a motion picture camera, Ballanger et ale (1966) reported that it was possible to determine which of the parameters of ciliary activity was affected, (i.e., force, amplitude, frequency' of the metachronal wave length or velocity). Another study, published by Rylander (1966), was concerned with the 'mucus transport rate studied by direct microscopic obse~vatipn employing diffuse vertical illumination which fell on the ~ epithelial surface at different angles. The time required for particles such as aoot, seeds, or small pieces of metal to move a certain distance was noted. He stated, ho~ever, 26 I that Whether or not a~rest of the eiliary beat always causes the mucus flow to stop remained to be proven. Hoorn and Tyrrell (1966) inoculated re~piratory syncy- tial eRS) virus in adult tracheal cultures from ferret and human embryo. tract They found that,all th. upper respiratory ~oviru8es tested would multiply in organ cultures of human embr,yo and ferret but not all would produce damage to the cilia. Influenza A and B caused cell destruction but the parainfluenza viruaes, influenza C and respiratory syncytial virus produced no obv1ous damage to the cells. They alao noted that cultures which .howed normal ciliary activity while supporting the growth of one virus may be able to produce only one percent of the normal yield of a second virua. They concluded that organ cultures showed many of the specific character11tics of the intact host, in that ciliated epithelium from different apecies is morphologically Similar but lu.epti~le to different viruses. Craighead (1966) experimented with parainfluenza type 3 virus in guinea p1q nasal mucosa anc! found that the virus J production was not affacte4 bY the immune status of the animal u.ed aa a .ouree of tis.ue. The virus grew wall in the organ culture but pro4uced 11ttle if any cellular damage. He o~.rve4 fluieS. that interferon wal, net detected in the culture He po1nted out that the mUCU8 layer of the mucosa was often lo.t durin; preparation thU8 the organ cultures 4iffered from intact r •• p,1ratory tract tis.ue in that regard. 27 McIntosh et al. (1967) reported that when they employed ordinary tissue culture techniques only 20-35% of adults with acute upper respiratory tract infections yielded viruses.," With the use of human embryonic tracheal organ cultures however, they were able to isolate viruses from one-third of 23 nasal washings which had failed to yield viruses by standard tistlue cultu;:~~c techniques. Of these isolates several could only be detected by their ciliary immobilizing effect in organ culture. It has been shown that interferon is formed in the respiratory tract in response to virus infection and interferon has also been found in fluids of organ cultures of mouse, calf and pig tJ;"achea infected with Sendai virus (Hoorn and Tyrrell, 1969). Finter (1968) showed that mouse brain interferon protects mouse trachea against influenza virus infection. Hoorn (1966) reported that human tracheal organ cultures were more sensitive than monkey kidney tissue cultures or embryonated e9gs for the isolation of an influenza A virus. Collier (1969) reported that when lOS colony forming units of 11¥coplasma pneumoniae were inoculated into hamster tracheal organ cultures, ciliary activity ceased in 48 to 72 hours and that the time required was dose dependent. He observed that the pathological changes observed were not seen in tracheas infected with avirulent M. pneumoniae suggesting that this was a good model for analysis of 28 pathogenic mechanisms. From this literature survey it seems certain that virus-mycoplasma interactions do occur. The purpose of this study is to investigate the possibility that a virusmycoplasma interaction may cause increased host damage as suggested by the observations made of infections in tracheal organ culture. MATERIALS AND METHODS I. PREPARATION OF ORGAN CULTURES The preparation of the organ cultures was adapted from the technique of Hoorn and Tyrrell (1965). Five week old Swiss-Webster mice from a colony found previously to be free of mycoplasma were used. The tracheas from just be- low the larynx to just above the carina were excised in bloc by sterile' technique and stripped of excess connective tissue. The tracheas were placed in Hanks balanced salt solution plus 1000 units per ml of penicillin G and allowed to soak for 30 minutes. After soaking, the tracheas were placed on dissecting wax and cut with razor blades held with hemostats to avoid damage to the epithelium. The tracheas were cut either into rings 1 mm in thickness or cut into two longitudinal strips. The longitudinal strips were then cut into squares measuring about 2 mm'on an edge. Each trachea yielded from 8 to 10 rings or 8 to 10 squares of tissue. Traeheal tissue was then placed, with the aid of an inoculating needle, into 16 x 150 mm roller tubes in such a way so that the tissue would adhere to the glass surface. The use of roller tubes for organ culture was reported by Harnett and Hooper (1965). Each roller tube culture contained the tracheas from three mice yielding 30 approximately 30 pieces of tissue. The ciliary action of the epithelial cells was best observed with a Zeiss Universal Microscope employing 63 x or 160 x total magnification. Only explants showing strong ciliary activity were retained for experiments. The pre- sence or absence of ciliary activity and of gross alterations of the structure of the tracheal mucosa was observed and recorded. II. MYCOPLASMA The mycoplasma used in this study was ~. pulmonis isolated from an infected rat in the laboratory of Dr. B. C. Cole, university of Utah. The mycoplasma pool was prepared by growing the organism in broth medium. The broth medium used was that of Chanock et ale (1962) and consisted of seven parts Difco PPLO broth, two parts horse serum (unheated) and one part yeast extract (Microbiological Associates, Inc.) plus 1000 units penicillin G per ml. III. VIRUS The PR-8/34 strain of influenza A virus used in this study was obtained from the Research Reference Reagent Laboratory of the National Institute of Health, Bethesda, Maryland. The virus had a passage history of 8 passages in ferrets, 593 passages in mice and 168 passages in chick embryos before it was received in this laboratory. The virus 31 pool was prepared in this laboratory by passage in the allantoic cavity of embryonated eggs according to the method of Hirst (1942). The seed virus was allantoic fluid stored at -70 C and diluted for use with gro~~h medium. IV. CULTURE MEDIUM The culture medium for the tracheal organ culture found previously to be suitable for this work, consisted of L-15 growth medium (Microbiological ASSOCiates, Inc.) described by Leibovitz, (1963) plus 100 units per ml of penicillin G and 0.2% bovine serum albumin (Microbiological ASSOCiates, Inc.). v. ~OPLASMA QUANTITATION The method for enumeration of viable tl. pulmon1s was based on the procedure of Miles and Misra (1938). Three 0.01 ml droplets of each serial tenfold dilution of tracheal organ culture supernatant flu1(l diluted in PPLO broth were placed on quadrants of PPLO agar (Chanock's medium plus 2% agar) plates on 100 mm diameter. The plates were allowed to stand closed at room temperature until the droplets had been absorbed thus preventing coalescence of the droplets on the plates. The plate cultures were then sealed with rubber dish seals (van Waters and Rogers, IDC., Salt Lake City, Utah) and incubated aerobically at 37 C. 32 The colonies were large enough to count after six days incubation. A stereoscopic dissecting microscope with a 15 x magnification (Bausch and Lomb, Inc., Rochester, New York) was used to view the colonies. VI. VIRUS QUANTITATION The virus samples were assayed using 50% end-point titrations in continuous lines of ei.ther BHK-2l (ba~ hamster kidney cells) or African green monkey kidney cells (vero). Evidence of virus infection was obtained by demonstrating hemadsorption with a 0.4% suspension of guinea pig erythrocytes. The technique for calculating the tissue culture infective dose (TC1D50) was that of Reed and Muench (1938). The hemadsorption test was based on that of Blair et ale (1970) and was performed as follows. The tracheal organ culture fluid was inoculated as 10-fo1d dilutions into the stationary tube tissue cultures and incubated at 33 C for 48 hours. After incubation, the cell culture medium was decanted and 0.2 ml of the guinea pig erythrocyte suspension was added to each tube. The tubes were incubated at 25 C for 45 minutes after which they were washed twice with cold buffered saline. The tubes were then read for hemadsorption of the RBC's to the infected cells. Adherence of the erythrocytes to the inoculated cells indicated virus infection. 33 VII. INOCOLATION OF ORGAN CULTURES Unless otherwise indicated, the organ cultures were inoculated on the second day of incubation designated day zero. The medium was removed and replaced with 1.0 ml of L-1S medium containing the virus or mycoplasma. The cul- tures were incubated at 33 C for periods up to lS days. The ciliary activity was assessed and recorded daily. The medium was completely removed every other day for virus or mycoplasma titrations. this time. A Fresh L-1S medium was replaced at piece of tracheal tissue was also removed every other day and fixed in formalin to be sectioned and stained at a later date. VIII. HISTOLOGY The methods for sectioning and staining the tracheal tissue was adapted from the procedures outlined in !2£ Histologic Technicians by Ann Preece. ~ ~~nual The tissue was first fixed in a 10% neutral formalin solution prepared by diluting 10 ml of a 37-40% formaldehyde in 90 ml of distilled water and adding a slight excess of CaCo3 • The formalin solution was at least 20 times the volume of the tissue. The tissue was then washed free of the formalin by dipping it in distilled water for 1.5 hours. Dehydration of the tissue was accomplished by 3 one hour washings in fresh 95% ethyl alcohol followed b¥ 3 one hour washings in 34 absolute alcohol. The tissue was cleared of alcohol placing it in cedarwood oil overnight. ~ The tissue was then washed in 2 changes of chloroform for 10 minutes each to remove the cedarwood oil. plished ~ The infiltration was accom- dipping the tissue in paraffin for 1 to 15 minutes then again for 1 to 2 hours. The tissue was then embedded in a solid mass of paraffin. The blocks were sectioned with a microtome yielding sections of from 6 to 10 microns in thickness. The sections were mounted on slides employing egg albumin as a fixative. DEMONSTRATION OF PEROXIDE PRODUCTION IX. The method employed to demonstrate peroxide secretion ~ H. pulmonis was that of Lind (1970). An agar square containing less than 25 mycoplasma colonies was placed on a glass slide. One drop of a 0.5% suspension of guinea pig erythrocytes in PBS pH 7.2 was placed on the agar block along with one drop of a 0.01% solution of methylene blue solution and allowed to react for 10 minutes. Some of the agar blocks were treated with 1000 units per ml catalase prior to the addition of the RBe's and the methylene blue. Dark blue staining erythrocytes next to the colonies constituted a positive test. The blocks were observed and photoqraphed in the microscope employing 160 x total magnification. 35 x. ELECTRON MICROSCOPY The procedure for the preparation of the tracheal tissue for examination with the electromicroscope was based on the technique described by Pease (1964). The mouse tracheal explants were placed in Millers buffer pH 7.1 overnight and then transferred to cold 3% glutaralde- hyde for primary fix.ation. 2~ osmium tetroxide. The tissue was postfixed in The samples were dehydrated in graded alcohols and embedded in epoxy resin (Epon 812). The embedded tissue was then sectioned with diamond knives on an MP-l Porter-Blum ultramicrotome, placed on grids and stained with aqueous uranyl acetate and Reynolds' lead Citrate. (1963). Reynolds· lead Citrate was described ~ Reynolds The sections were examined and photographed em- ploying a Zeiss EM 9 A electron microscope at magnifications ranging from 1650 to 20,000. XI. FLUORESCENT-ANTIBODY STUDIES The infected tracheal explants were first embedded 1n paraffin according to the method of Heron (1970). tracheal tissue was placed in 95% ethanol at 4 per10d of at least 18 hours for fixation. C The for a The tissue was then placed in cold 99% ethanol for 3 hours, passed through 2 xylol baths at 4 C for 3 hours and then placed in paraffin at 58 C overnight. The next day the paraffin block was 36 sectioned and the sections were deparaffinlzed in the following manner. The sections were placed on de-greased slides and passed through two changes of xylol at 4 C for 60 seconds for each change. The slides were then passed through two changes of cold 99% ethanol for 60 seconds followed by washes in 96% cold ethanol (10 sec.), 70% cold ethanol (10 sec.) and finally in 3 changes of phosphate buffered saline (PBS) at room temperature. After a1r- drying the specimens were stained by a method based on the procedure of Goldman (1968). The trachea infected with ~. pulmonis was stained the direct fluorescent-antibody technique. ~ The slides were fixed in cold acetone for 10 minutes and allowed to air-dry. The slides were then flooded with a 1:40 dilution of fluorescein isothiocyanate (FITC) conjugated mule anti- M. pulmonis (ASH strain) globulin Lot #7041527 (Baltimore Biological Laboratories, Baltimore, Maryland) or a 1:40 dilution of FITC conjugated normal mule globulin (BEL). After a 30 minute incubation at 37 C, the slides were rinsed in 3 changes of PBS, once in distilled water and allowed to air dry. Finally the slides were countersta1ned with 0.05% Evans blue for 5 minutes and mounted with coverslips employing phosphate buffered glycerol. The influenza virus infected tissue was stained indirect technique. ~ After fixing the tissue in acetone, the 37 the slides were flooded with a 1:10 dilution of chicken anti-influenza A PR-8/34 immune serum or normal chicken serum (Research Reference Reagent Laboratory, NIH) and incubated in a moist chamber at 37 C for 1 hour. The slides were then rinsed in distilled water and allowed to air-dry. After drying the slides were flooded with horse anti-chicken FITC conjugated globulin Lot #9013 (progressive Laboratories, Inc., Baltimore, Maryland) and incubated at 37 C in a moist chamber for 30 minutes. Finally the slides were rinsed in 3 changes of PBS, once in distilled water, counterstained with 0.01% Evans' blue and mounted. The controls included infected and uninfected tracheal tissue stained with specific and normal conjugated mule and horse globulin. The slides were examined with a Ziesa Universal fluorescent microscope equipped with a Osram 200 watt mercury burner employing a BG-12 exciter filter and a No. 42 barrier filter. The FA tests were considered valid if no fluorescence was observed with the controls consisting of uninfected tissue or infected tissue stained with normal conjugated globulin and if fluorescence was observed with infected tissue stained with specific conjugated antiserum. RESULTS I. MYCOPLASMA PULMONIS IN MOUSE TRACHEAL ORGAN CULTURE A. Growth of M. Pulmonis The mycoplasma inoculum was 104 • 1 CPU per ml in each of three culture tubes. The medium was removed, ex- plants were washed with fresh medium, and new medium was replaced at 48 hour intervals. moved was titered for in Table I. ~. The supernatant medium re- pulmonis and the titers are shown Growth of the mycoplasma occurred during the 15 day test period as indicated by a total production of H. pulmonis of 106 • 4 CPU above the amount of organisms originally introduced into the organ cultures on day zero. Ex- plants showing complete inhibition of ciliary activity were first noted on day 9 and increased to include 67% of the tissue explants by day 15. The control cultures showed signs of slowing down of the frequency of ciliary beat but none of the tissue explants had completely lost its ciliary activity during the 15 day test period. B. Histopathology The histological structure of tissue from uninocu- lated control cultures was well maintained through day 15. Figure 1 shows that by day 5 the trachea exposed to the 39 Table I MzQOp~a@~ Rylmonls Infection of Mouse rae ea1 Explints in Oulture fest 0 1 Dati E2st1nocu1atlo~ 3 5 7 9 11 13 15 MYcoplasma infected cultures flter a ft·1 5.7 6.3 5.4 4.9 4.8 4.3 4.2 3.6 0 Oiliary 0 6 N 0 0 0 0 0 9 act1vity TC 14 14 14 14 14 14 14 14 14 4.7 6.3 4.3 4.8 N 0 0 0 0 0 T 16 15 15 15 15 4.1 T1ter Oiliary N 0 activity T 19 5.0 6.2 4.9 0 0 0 19 17 16 0 19 16 1 16 5 16 12 16 0 0 0 0 2 7 38 67 4.7 4.3 4.2 3.7 f1ter 011iary activity Percent 401 Nd 0 Mean titer 401 5.1 6.3 4.8 4.9 1 15 3.3 3.6 6 2 15 15 3.7 9 15 4.7 4.5 4.4 4.8 3.7 0 4.8 Uninfected oultures Oiliary actiVity N0 T 14 0 14 0 0 14 13 12 12 12 12 12 Percent N 0 0 0 0 0 0 0 0 0 0 0 0 0 0 LoglO OFU per ml ~. pulmon1i yield from supernatant medium entirely removed every 48 hours. b Number of tracheal explants showing no oiliary activ1ty. c Number of tracheal explants soored for oiliary activit)"_ d Percent of total explants showing no aotiv1ty. a 40 B Figure 1. Appearance of mouse tracheal explants after 5 dals incubation. (A) Infeoted with H. ~UlmOn1S. (B) Uninfected oontrol. H&E stain X 40 • 41 H. pulmonis had started to show evidence of damage to the epithelial layer. The epithelial architecture was startinq to become disorganized. The nuclei no longer were present at the base of the cells but had started to migrate toward the apex. The nuclei also appeared to have shrunken and become thickened. Cytoplasmic vacuolization was also apparent in some of the cells. By d~ 11 the epithelial organization was completely lost in most cases in cultures containing the mycoplasma. Damage to the epithelium seen in the Hand E stained preparations correlated well with the reduction and cessation of ciliary activity. C. Immunofluorescence A fluorescent-antibody study was conducted to determine whether or not the mycoplasma organisms were associated with the cells and if so, whether the mycoplasma were invading the cells or residing on the cell surfaces. The infected tracheal sections after 5 days in culture when stained with specific fluorescent-antibody showed a spotty discontinuous bright line of fluorescence along the surface of the ciliated epithelium. There was no fluores- cense within the epithelial cells or in the submucosal connective tissue. The tissue stained by fluorescent- antibody (FA) after 11 days in culture exhibited a more intense and continuous fluorescence along the epithelial surface. Some small scattered areas of bright fluorescence 42 were also observed in the submucosal loose connective tissue. No fluorescence was observed within the cells themselves. No specific fluorescence was detected in control preparations. D. Electron MicroscoPl Infected explants were examined with the elec- tron microscope to determine the site of mycoplasma association with the tissue. Possible cell reaction to the mycoplasma was also looked for. Figure 2 shows an electron photomicrograph of a normal mouse tracheal epithelial cell from an explant after culturing for 1 day. There are many ci11a present which are seen in longitudinal and transverse section. The presence of numerous mito- chondria is characteristic of ciliated cells. Tonofila- ments and granular endoplasmic reticulum were also observed. Figure 3 shows an electron-photomicrograph of tracheal tissue after culturing in the presence of pulmonis for 7 days. H. The absence of cilia and the presence of numerous mycoplasma on the surface of the epithelial cells is evident. ing goblet cell. The cellon the right is a mucus secretThe electron-dense cytoplasm is due to the presence of densely packed ribosomes. granules are present in the cytoplasm. Many large mucus The mycoplasma on the surface of the goblet: cell appear to be sitting in cup-like depressions in the cell membrane indicating that 43 Figure 2. Eleotron micrograph of normal mouse tracheal ex~lant after 1 day in oulture. Note cilia (0) mitochondria (M) and microvilli (Mv) X 15,000. 44 Figure 3. Electron miorograph of M. pUlmo~s infeoted mouse tracheal explant after 7 ~ys in culture. Note myooplasma eM) and mucus granules eMu) x 15,000. 45 the cell may be responding to the presence of the mycoplasma. There was no indication of fusion of the myco- plasma membrane with that of the cell or active engulfment of the organism by the cell. It was evident that there was a close association of the mycoplasma with the cell membrane. No mycoplasma could be recognized within the cells, however, bodies resembling mycoplasma were seen in the submucosal connective tissue in specimens from cultures after 11 days. A small degree of cytoplasmic vacuolization was seen in cells from explants after S days of infection. ~ day 11 many more vacuoles were seen. Mycoplasma organisms were never seen within the vacuoles, however. H. pulmonis organisms were frequently s'een around the bases of cilia but never appeared to attach to the cilia. I. Effect of Receptor Destroying Enzyme Since the mycoplasma appear to attach to the cell surfaces, an experiment was conducted to determine whether or not receptor destroying enzyme (RDE) might effect the attachment and growth of the mycoplasma and its subsequent effect on the ciliary activity of the cells. Two tubes containing tracheal tissue were treated with receptor destroying enzyme (ROE) (Microbioloqical Association, Inc., Albany, California) containing 2S units per ml in PBS plus 0.1% C~C12. The tracheal cultures were 46 treated for a period of one hour at 37 C in a roller drum. The tiesue was then washed three times in PBS, then 1 ml of growth medium (previously described) containing the mycoplasma was added to the cultures. cultures not treated with RDE Controls consisted of but inoculated with organisms and treated cultures not inoculated with organisms. The data 1n Table II and Figure 4 indicate that the treatment with H. had no apparent effect on the yield of pulmonis from the culture supernates. When 104 • 4 colony RDE forming units (CFU) of the mycoplasma were inoculated into RDE treated and control cultures the total mycoplasma production was not significantly different. Table I I indicates that the ciliary activity was apparently affected to the same degree in the infected cultures. RDE treated and untreated mycoplasma The ciliary activity of the RDE treated control cultures containing no organisms was not significantly different from that of the untreated control cultures indicating that the RDE was not toxic to the tracheal tissue. P. Effect of Viable and Heat Treated Tissue On Growth of M. Palmanis The mycoplasma appear to grow in close proximity to the tracheal epithelial cells thus they may require viable tracheal cells for their growth. The following experiment was designed to answer this question. 47 Table II Effect of Reoeptor Destroying Enzyme (RDE) on Myooplasma Ru1moQ!Ainteotionot Mouse Tracheal Exp1ants 1 DalS post!noculat1on 11 5 7 3 9 Titera b 4 • 4 No O.A. 0 'fotale 10 4.7 4.9 5.2 5.3 4.9 4.6 10 10 4.6 4.6 RDE 4.4 T1ter No C.A. 0 10 Total 0 0 Org. No No C.A.. RDE No No C.A. Total 0 11 No O.A. 0 !fest Org. plus RDE Org. plus RDE Org. No iDI No Org. s b c 0 Titer fotal Total 0 10 0 10 0 10 0 10 5.4 5.1 5.7 0 4.6 0 0 2 9 9 9 9 0 10 1.0 0 4.4 4.5 4.6 4.9 5.3 5.5 5.2 0 0 0 1 2 0 1 12 12 12 12 12 12 12 11 0 11 0 11 0 0 11 11 13 15 3.9 3 10 4.2 4 10 3.8 3.5 3 9 4 9 4.8 3.6 4 4 12 12 0 0 0 0 9 0 9 0 9 9 9 9 0 0 0 0 9 0 0 11 10 10 9 9 LOglO OFU per m1 !. pu~monis y1eld trom supernatant med um entirely remove every 48 hours. Number of tracheal explants showing no ciliary activl ty. Number of tracheal exp1ants scored for oiliary aot1vl ty. 48 Loglo Ru1mon1s with RDI oru pulmonis without RDE per ml o Figure 4. 1 3 5 7 Days 9 11 13 15 Yield of !. pulmonis from supernatant medium of mouse tracheal organ oultures treated with reoeptor destroying enzyme (RDE) • 49 Three culture tubes containing tracheal tissue were heated at 60 C for 30 minutes. These cultures together with cultures containing viable tissue were inoculated with 103 • 1 CPU of H. pulmonis and incubated for a period of 15 days. The entire medium was removed and replaced at 48 hour intervals. The medium removed from the cultures was titered for mycoplasma at this time. A controlcul- ture containing medium but no tracheal tissue was also inoculated. Table III and Figure 5 show that the myco- plasma grew well in the cultures containing viable tissue but the cultures containing heat treated tissue failed to support the growth of the ~. pulmonis. The presence of organisms on day three in the cultures containing heat treated tissue probably indicates that the organisms were caught or trapped in the crevices of the tracheal tissue and released later rather than any growth actually taking place. G. Effect of Medium Supplements H. pulmonis appears to require viable tissue for growth in L-15 growth medium. An experiment was desiqned to establish whether or not the viable tissue could placed ~ a medium supplement. be re- Nine culture tubes of heat treated mouse tracheal explants were inoculated with 103 • 4 CFU of ~. pulmonis in L-1S growth medium (previously described). Two tubes had 0.02 mg per ml of cholesterol 50 Table III MYooKlasma ¥ulmon1sin Organ Oultures of V1able an Heatreated Mouse Tracheal Etplants Test a 1 RIll iost~noQulat,on :5 5 7 9 11 13 15 4.5 4.5 3.9 Vtable Explants a 'r1terb ;.1 2.4 4.5 Titer 3.1 2.5 4.6 4.7 5.5 4.9 4.8 4.6 4.1 Titer 3.1 2.5 4.5 4.6 5.4 5.0 4.8 4.7 3.9 4.8 5.3 4.5 Heat treated explants C Titer 3.1 2.6 1.7 <1.5 <1.5 <1.5 <1.5 <1.5 <1.5 T1ter 3.1 2.2 1.5 <1.5 <1.5 <1.5 <1.5<1.5 <1.5 Titer 3.1 2.1 1.8 <1.5 <1.5 <1.5 <1.5 <1.5 <1.5 Oontrol Titer a Organ cultures containing viable tracheal tissue. b LoglO CFU per ml c ~. pulmonis 1n supernatant fluid. Organ cultrues contain1ng heat treated tracheal tissue. d Control cultures containing growth medlumbut no traoheal explants. 51 r=:J I· pulmonis with viable explants V'7I 'll. rulmoW. s wi th heat treated ~ exp ants Loglo OPU per ml t::I 6 !. pulmonis with no explants 5 4 , 2 1 o Figure 5. o 1 3 5 7 Days 9 11 15 Yield of M. ~ulmonis from the supernatant medium ot-cultures containing viable and heat treated mouse tracheal explants. Broken line indicates limit ot sens1tivity ot the assay system. S2 I added, two tubes had 10% yeast extract I had 0.02 mq per ml of cholesterol added, two tubes had , 10% yeast extract added, two tubes had 0.02 mq per ml add~d, two tubes cholesterol plus 10% yeast extract added, two tubes had 5% horse serum added, one control tube had L-15 medium plus dead explants and the last control had L-15 medium and mycoplasma without any tissue. The medium was com- pletely removed every other day and replaced with fresh medium without washing. The medium removed was titered for mycoplasma and the results expressed as mean titers of two cultures are presented in Table IV and Figure 6. The greatest degree of growth occurred in cultures containing added cholesterol plus yeast extract or added 5% horse serum. Tubes containing only added cholesterol also supported the growth of mycoplasma, but not to the same degree. Cultures with added yeast extract or no added supplements did not promote the growth of the mycoplasma. H. Infected Culture Filtrate Effect on Ciliary Activity This experiment was designed to determine if ciliary inhibition caused by the mycoplasma required the presence of actively metabolizing organisms. Media was removed every other day from 3 cultures originally infected with 104 • 2 CFU of ~. pulmonis. This medium was filtered through 0.22 micron mean pore size Millipore filters 53 Table IV Effeot of Med1um Supplements on The Yield of MYooplasma pulmonis From The Supernatant Medium of Heat Treated Traoheal Exp1ants Supplement a o 1 3.4 3.6 DalS postinoculation 3 5 7 9 11 13 15 Cholesterol Titer b 3.6 3.9 3.3 4.6 4.3 4.6 4.7 3.5 3.3 5.8 5.6 6.4 6.8 6.7 5.6 5.9 6.2 5.7 5.6 5.4 6.1 Yeast extraot Titer Yeast extract and oholesterol Titer 3.4 2.8 5 peroent horse serum Titer 3.4 4.8 1-15 medium only Titer 3.4 2.1 (1.5 (1.5 (1.5 (1.5 (1.5 (1.5 (1.5 Heat treated t1ssue plus L-15 Titer a 3.4 2.3 (1.5 (1.5 (1.5 <1.5 (1.5 (1.5 (1.5 Supplements added to L-15 growth medium in oultures of heat treated mouse traoheal explants. b Logl O eFUper ml ~. Rulmon1s mean t1ter ot ~uper­ natant fluid from two cultures. 54 t::J Cholesterol ~ Yeast extract plus cholesterol Log lO OPU per ml • Horse serum 6 5 4 0 Log lo OFU per ml Yoast extraot ~ L-15 medium alone 1 3 :5 2 1 0 0 'igure 6. 5 7 Days 9 11 15 Effeot ot medium supplements on the y1eld of H. pulmon1s from supernatant med1um of heat treated mouse traoheal explants. Broken l1ne ind10ates limit of sensit1vity of the assay system. 55 (Millipore Corp., Bedford, MaSs.) and treated with 12 micrograms per ml Tylosine (Flow Laboratories, Rockville, to inhibit mycoplasmal growth. M~.) This treated medium was used as a replacement medium for 3 un1nfected cultures. The treated medium was assayed after each treatment for m¥coplasma and was found to be free. Controls consisted of un1nfected cultures with fresh growth medium replacements and cultures containing filtered tylosine treated medium from uninfected cultures. appear in Table V. The results of the experiment In the cultures containing actively metabolizing mycoplasma the ciliary activity progressively deteriorated until the end of the experiment on day 15 when 50% of the tracheal explants had lost all ciliary activity. The cultures with treated medium from infected cultures exhibited ciliary activity not significantly different from the control cultures. It appears that the effect of mycoplasma on the ciliary activity was not due to the release of stable toxins or depletion of an essen- tial nutrient in the medium b¥ the mycoplasma since the treated medium from the infected cultures did not produce ciliary inhibition in the mycoplasma free cultures. I. Demonstration of Hydrogen Peroxide Production When agar blocks containing colonies of ~. pulJDonis were treated as described previously in materials and methods, the erythrocytes that were in contact with the 56 Table V Effeot of sterile Oulture Filtrates Jrom !. pulmonis Infeoted Oultures on Ciliary Activity of Unintected Mouse Traoheal Explants 0 Teat Infected explants Na 0 01l1ary Tb 24 activ1ty %NC 0 1 0 24 0 DalS postinoculat!on 11 7 9 3 5 1 ' 24 0 13 15 11 24 12 24 50 1 24 0 4 24 17 5 24 21 9 24 0 0 25 0 25 1 1 0 0 4- 4 0 15 0 0 15 0 0 15 0 0 15 0 0 0 14 0 14 14 0 0 ;8 46 Sterile filtrates on exp1ants 011iary activ1ty 0 T 25 N %N 0 0 25 0 0 25 0 25 0 25 25 2 25 8 Un1ntected oontrol explants C11iary activity N 0 T 15 %1 0 0 15 0 0 15 0 0 0 15 0 15 0 0 1 14 0 Media trom control on exp1ants C111ary activ1ty N l' ~N 0 14 0 0 14 0 0 14 0 14 0 a 0 14 0 a Humber of explants showing no oiliary aotivity. b Number of exp1ants soored for cil1ary activity. 0 Percent of total explants showing no activity. 57 colon1es stained a deep blue color as shown in Figure 7 indicating the mycoplasma colonies were producing hydrogen peroxide. If the colonies were pretreated with catalase the deep blue color of the erythrocytes did not occur. Colonies of MYcoplasma salivarium known not to produce hydrogen peroxide also failed to elicit the reaction when treated with erythrocytes and methylene blue. The results of this experiment indicated that colonies of the M. pulmanis li'berate hydrogen peroxide. The mechanism for the reaction as explained b¥ Lind (1970) is as follows: Normal erythrocytes accumulate methylene blue which they convert to a colorless leukomethylene blue. The mainten- ance of the methylene blue 1n the reduced state is dependent upon enzyme functions of the erythrocyte. The high concentration of hydrogen peroxide interferes with the enzyme function allowing the methylene blue to become oxidized thus resulting in the deep blue color. J. Effect of Catalase The previous experiment demonstrated that palmonis does produce hydrogen peroxide. ~. If production of hydrogen peroxide is responsible for the inhibition of ciliary activity, then exogenous catalase added to the infected cultures should reduce the inhibitory effect. Three tracheal organ cultures were inoculated with 104 • 5 CFU of ~. pulmonis and catalase ,(Sigma Chemical Co., st. Louis, Figure 7. Methylene blue staining ot erythrooytes (arrows) surrounding a oolony of ~. Rulmonls indioating hydrogen peroxide produot1on. Magnification 160 x. 59 Mo.) in a concentration of 0.03 rog per rol was added to the growth medium. The added catalase should destroy any hydrogen peroxide produced by the M. pulmon1s. Glucose in a concentration of 1% was added to 3 separate organ culture tubes without catalase. Cohen and Somerson (1969) reported that glucose will stimulate peroxide production by mycoplasma. Three infected cultures were also incu- bated in the presence of 3.5 mg per ml 3-amino-l,2,4-triazole (schwarZ-Mann, Orangeburg, N. Y.) which was reported by Cherry and Taylor-Robinson (1970a) to inhibit any catalase that may be produced by the explants. Controls consisted of uninfected tracheal cultures with added catalase or added amino-tr1azole. ~~coplasma titrations and observations of ciliary activity were conducted every other day at which time the medium was replaced. The results of this study are presented in Table VI and Figure 8. The total yield of the mycoplasma cultured under the different conditions did not differ significantly. The ciliary activity, however was inhibited to a greater degree in the cultures that did not contain added catalase. After 15 days, the number of tissue explants showing complete cil10stasis in the catalase containing cultures were less than half that of the tissues from cultures without added catalase. The cultures with added glucose or amino- triazole did not demonstrate increased ci110stasis over that of the infected cultures without additives. The 60 Table VI Effeot of Added Catalase, Glucose, and Amlno-1'r1azole on HYooRlasm~ 2ulmonis Infeotion of Mouse Traoheal Organ Cultures Supplement 0 1'i ter~ 4.5 4.5 N 0 0 38 38 No supplement TO %N Glucose T1ter N T %N Titer triazo1e Catalase No Organisms A.m1notriazole No org. N T %N N T %N N T %N 13 15 4.8 5 38 13 4.2 9 38 24 486 5.4 7 48 15 4.7 17 48 35 4.9 23 48 47 4.7 0 38 0 5.3 4.9 5.0 5.1 4.6 6 4.7 8 0 0 38 0 38 38 16 38 21 5.2 5.2 4.9 4.7 17 35 4.8 0 38 4.R 4.9 0 0 38 0 38 5.1 0 0 0 0 4.5 4.2 4.7 0 48 0 Titer 4.5 0 Oatalase N l' 39 %N 0 Ami no- 1 DayS Eost~noculat1on 11 9 3 5 7 0 48 0 4.6 0 39 0 0 48 0 48 0 0 38 0 5.2 1 5.2 4 38 10 4.8 48 6 48 0 13 0 0 4.5 0 35 4.5 0 35 0 4.6 0 35 4.9 1 5.0 0 0 35 35 6 0 15 0 15 0 0 0 15 0 0 15 0 0 0 0 0 12 0 12 0 12 0 12 0 12 0 0 0 0 0 0 0 2 15 1 38 2 15 38 40 6 10 35 11 35 17 29 0 15 0 15 0 0 15 0 15 0 12 0 12 0 12 12 0 0 0 0 4 35 48 0 0 b Number of explants showing no ciliary aotivity. c Total number of explants scored for ciliary act1vity. d Peroent of explants showing no cil1ary activity. a Log lO CFU per ml of M. Eulmon1s, mean titer of :3 tubes. 61 Percent absent oiliary aotivity 100 80 60 o Figure 8. 1 3 -M. Rulmonis no supplements 0 0 6" 6 C D Gluoose • • • Amlno-triazol 5 6 7 Days Oatalase Un1nfeoted control 9 11 13 15 Effect of added catalase, gluoose, and am1no-trlazole on the oiliary activ1ty in ~. pulmonls.1nfeoted tracheal organ oultures. 62 uninfected controls did not show significant ciliary inhibition indicating that the catalase and amino-triozole were not toxic to the tracheal explants in the concentrations employed. production ~ the It appears then that hydrogen peroxide ~. pulmonis when closely associated with the cells may be responsible fox the ciliary inhibitory effect. II. INFLUENZA A VIRUS IN MOUSE TR.1i,CHEAL ORGAN CULTURE A. Growth of Influenza A (PR-§l Table VII shows the production of the virus in the tracheal organ culture. The virus inoculum was 103 • 3 TOIDSO per ml in each of three cultures. The entire medium was removed, the explants washed, and fresh medium was replaced at 48 hour intervals. The removed supernatant medium was t1tered for virus. The mean total amount of virus produced over the 15 day period was 10 3 • 7 TCIDSO. Control tubes containing 10 3 • 3 TCIDSO influenza virus in growth medium without tracheal explants were also included. The infectious virus did not survive in the controls for 48 hours. on day zero tracheal tissue in the roller tube cultures was examined and the tissue explants showing the greatest degree of ciliary activity were marked by circling the tissue with a diamond stylus. These pieces of tissue were monitored every other day at which time the ciliary activity was 63 Table VII Influenza A CPR-8) Virus in Mouse Tracheal Organ Oulture Test 0 1 Data Eo~tinooulat~on 11 7 9 3 5 Virus infected Cultures Ti tera b3 • 3 2.8 3.3 0 0 Oiliary NC 0 16 actlvi ty T 17 16 2.8 Titer Ciliary act1vity Titer Oiliary activity N T Mean titer Percent T 2.3 1.5 13 15 15 15 2.5 2.5 16 16 15 15 2.3 1 15 2.5 1.8 1.8 1.5 14 14 5 5 15 15 15 15 3.0 2.3 0 18 9 18 2.; 15 18 2.5 16 18 2.5 2.8 2.3 0 0 16 0 16 ;15 15 2.8 1.5 2.5 2 4 6 20 18 18 2.8 3 18 3.3 2.6 2.8 2.5 2.7 2.7 2.2 2.1 1.8 0 0 12 27 42 87 94 3.3 N 15 2.3 0 3.3 0 13 0 20 Nd 0 0 0 0 4 Virus in cultures without explants Titer 3.3 0.2 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 Uninfected control cultures Oiliary act1vity N 0 T 14 0 14 0 13 0 13 0 0 13 13 0 12 0 12 0 12 b Log10 TCID~o per ml influenza A virus yield from supernatan med1um entirely removed every 48 hours. Number of traoheal explants showing no ciliary activ1ty. c Number of tracheal explants scored for oiliary activity. d Percent of total explants shanng no aotivity. a 64 scored as either present or absent. Table VII shows that by day seven 12% of the tissue scored showed no ciliary activity. Tissue explants sho\d.ng complete ciliostasis increased 1n number until day 15 when 94% of the tissue B.Y explants being scored exhibited no ciliary activity. day 15 the uninfectcd c~ntro1 tj~GUC explants showed com- plete loss of ciliary activity. B. Hist0p!thol~ The histological sections of infected tissue revealed progressing damage to the epithelial layer which was first evident on day 3. Figure 9 shows the presence of pyknotic nuclei and flattened appearance of the epithelium. cells. No cilia can be seen on the surface of the The control however, shows typical pseudostrati- fied columnar cells with cilia. After 11 days the epithe- lial layer has been completely destroyed. de~is Only necrotic could be seen where the epithelium once was. The control tissue explants showed evidence of some reorientation of the nuclei but otherwise appeared healthy. c. Immunofluorescence Tracheal sections representing infection for 5 and 11 days were examined for evidence of fluorescence after staining with specific fluorescent-antibody against influenza A virus. ~ day 5 the majority of the epithelial 65 B Figure 9. Appearanoe ot mouse tracheal explants after 5 days 1noubation. (A) Infected with influenza A virus. (B) Uninfected control. H&E stain X 400. 66 cells showed bright diffuse fluorescence shown in Figure 10. The fluorescence appeared to be confined to the cyto- plasm. Weak fluorescence was seen in the submucosal area but probably represented background autofluorescence. By day 11 the fluorescence was very spotty and appeared to be confined to clumps of degenerate cells still clinginq to the basement membrane. specific fluorescence. Control preparations showed no The immunofluorescent studies . indicated that the influenza virus ~eplication was confined to the epithelial cells. D. Electron Microscopx Figure 11 shows the infected tracheal tissue after 3 days. Virus particles can be seen adhering to the cilia. It appears as though the virus particles were acting as a bridge causing the cilia to clump together. This sticking together of the cilia was noted in all the electron photomicrographs of the virus infected tissue. Ciliary clumping was not noted in any of the controls or mycoplasma infected tissue. The oval structures near the surface of the cell membrane are cross-sections of microvilli. Evidence of viral inclusions in the cell cytoplasm were not seen. structures resembling microvilli were seen that may have been Virions budding off of the cell membranes. Fiqure 12 shows a closely packed cluster of virions attached to a cilium which can be seen in the center of the cluster. 67 Figure 10. Mouse traoheal explant infected in oulture with influenza ~ virus after 5 days incubation (Indirect immunofluoresoent technique). Speoific fluoresoenoe is limited to the epithelial cells. Magnification 160 x. 68 F1qure ~l. Electron micrograph of influenza A/PR-8 infected mouse tracheal explant after 3 days in culture. Note virus particles (V) and cilia (e) x 30,000. 69 Figure 12. Electron micrograph of a cluster of influenza A-PR-8 virus (v) attached to cilia (C) X 60,000. 70 E. Effect of Receptor DestroYing Enzyme Tubes containing tracheal tissue were treated with receptor destroying enzyme (RDE) (Microbiological Association, Inc., Albany, California) containing 25 units per ml in PBS plus 0.1% CaC1 2 • The tracheal cultures were tr.eated for a period of one I hour at 37 C on a roller drum. The tissue explants were then washed three times in PBS, then 1 ml of growth medium, previously described, containing the virus was added to the cultures. Controls consisted of cultures not treated with RDE but inoculated with organisms and treated uninoculated cultures. The culture medium was removed, the exp1ants washed, and fresh medium without RDE was replaced at 48 hour intervals. The removed medium was titered for virus. The data in Table VIII and Figure 13 shows that virus titers of the supernatant fluid were greatly reduced from day one through day 13 in the RDE treated cultures. When 103 • 8 TCIOSO of virus was inoculated into ROE treated cultures the titer fell to less than 0.15 TC10SO per m1 by day 7 and then remained low until day 15 when they were the same as those of the controls. The untreated control cul- tures supported the influenza virus growth well through the test period. The mean total virus produced in the RDE treated cultures was 102 • 2 TC1DSO compared with mean total virus production of 103 • 8 TCIDSO in the untreated cultures. 71 Table VIII Effect of Receptor Destroying Enzyme (RDE) on Infection by Influenza A in Mouse Tracheal Organ Oulture Test 0 1 DSl! 2ost1nooulatloA 11 7 9 5 3 13 15 Virus Titera b3• a 1.3 <0.1 0.8 <0'.;1 1.3 1.8 1.8 1.5 0 0 0 plus No O.A. 0 a 0 0 1 2 RDE Total C 16 16 14 14 13 13 13 13 13 3.8' 1.5 0.2 Virus Titer 0 0 plus No O.A.. 0 15 14 RDE Total 15 Virus Titer No No O.A. iDE Total RDE No 0 •.1. Total No Virus No RDE No Virus a No O.A. Total 3.8 2.5 2.8 <Cl~l <0.1 0 14 14 0 2.5 0 0 12 0 12 12 12 0 10 0 10 0 0 9 9 0 0 11 0 0 11 10 0 11 2.8 1.5 1.; 1.5 1.3 0 14 0 14 1 14 1 3.3 3.3 2.8 9 12 1.3 10 12 14 3 12 4 12 0 9 0 9 0 9 a 0 9 9 0 10 0 9 0 0 0 9 9 9 1 12 LoglO TOIDso per ml influenza A. b Number of traoheal exp'lants showing no oiliary activity. c Number of tracheal explants soored for oiliary activity. 72 Log lo TOID50 per ml ~ Influenza A virus plus RDE 6 r==J Influenza A virus alone 5 4 o 1 3 5 7 9 11 13 15 Days Figure 13. Effect of receptor destroying enzyme (&DE) on the y1eld of influenza A virus from the supernatant medium of mouse tracheal organ oultures. Broken line indioates l1mit of sens1tiv1ty of assay systemo ·73 This was a significant difference (5% significance level with the Wald-Wolfowitz runs test, Siegel, 1956). Table VIII also shows that the ciliary activity was less inhibited in the RDE treated cultures probably as a result of reduced viral replication. By day 9 no tracheal explants in the RDE treated cultures showed complete ciliary inhibition where 3 out of 12 explants in the infected cul.tures without ROE showed loss of ciliary activity. F. Combined Infection With M8 Pulmonis and Influenza ! To determine the effect of a mixed infection, mouse tracheal organ cultures were infected with both influenza A virus and H. pulmonis. In the first experiment 10 3 • 4 CFU of ~e pulmonie was inoculated into six tracheal cultures on day zero. Three tubes served as mycoplasma single infection controls. On day 3 the medium was removed and medium containing 103 TCIDSO of influenza A PR-8 virus was added to 3. tubes infected with the mycoplasma and 3 uninfected tubes for virus single infection controls. Titers for both organisms were determined at two day intervals, ciliary activity was assessed and tissue pieces were removed for preparation of histologic sections. The entire supernatant medium was removed for titration and fresh medium was replaced. results as shown in Table IX indicated that the gr~lth The of 74 Table IX J(yooplasnw: pulmonis Followed by Influenza A V1rus in Mouse Tracheal Organ Culture 0 Test Mean titer xa 3.4 DalS postinoculation :; 11 9 5 7 1 4.; 4.8 5.6 13 15 5.7 5.5 5.4- 5.3 4.9 Mean t1ter Mb 3.4 4.2 4.4 5.3 5.4 5.7 5.6 5~1 4.7 Mean t1ter VO Mean t1ter Vd 3.0 2.9 2., 2.4- 2.; 2.1 ;.0 2.0 1.4 0.7 1.0 0.3 0.1 2.3 Oiliary aot1v1ty e 0 0 0 0 0 0 4 40 49 Influenza A 0 alone 0 0 0 0 14 23 68 80 !I. i.,Ulmonis 0 0 0 0 15 21 19 31 52 Unlntected control 0 0 0 0 0 0 0 0 0 M. pu1monis - aione plus Influenza A a b Log lO OFU per ml M. pulmonis cultured alone. LoglO CFU per ml M. pulmonls cultured with influenza A. - - LoglO T01D50 per ml influenza A cultured alone. d Log lO TOIDSO per ml influenza A cultured 't11 th !. 2ulmonis. e Percent of tracheal explants showing no ciliary activity. c 75 ~. pulmonis was not effected by the presence of the virus since the total mycoplasma yield for the control cultuX'es and mixed infected cultures were similar. The growth of the virus however, was significantly (5% significance level) reduced when cultured in the mycoplasma infected cultures. The virus titer in the control culture supernates was well maintained through day 15 0 Figure 14 shows a graph of the growth curves for each organism when grown alone or in the presence of the other. The combined effect of the mycoplasma and virus on the ciliary activity is also shown in Table IX. By the 15th day the cultures containing only the virus had more tissue pieces showing complete ciliostasis than the cultures of the mycoplasma alone or the cultures of the mixed infections. The ciliary activity of the cultures of mycoplasma alone and the mixed infection do not significantly. diffe~ All the infected cultures sho'" a much greater degree of ciliary inhibition than the uninfected control cultures. A graph of the percent ciliary inhibi- tion under the different experimental conditions appears in Figure 15. The second experiment was designed to determine the effect on the tracheal explants and the growth of the organisms when the cultures were first infected with the virus followed in 3 days b¥ the ~. pulmonis. The influenza virus was added to 6 cultures on day zero in a concentration 76 H. pulmon1s DAlone ~Dual 1nfection LoglO oru per ml Influenza A virus o LoglO TOID50 per ml Alone . ~ Dual infeotion :; 2 1 o Figure 14. 1 :; 5 7 Days 9 11 13 Yield of !. pulmonls and influenza A v1rus from supernatant medium of dually infeoted mouse tracheal organ cultures when !. Rulm9Jtt! was inoculated first. Broken line indlcatco the limit of sensitivity of tho aSBay systemo 77 lercent absent ciliary aotivity 100 o F1gure 15. 1 3 alone 0 o !. 6 6 Influenza A alone 0 tJ M. • e 5 pu~monis PU~!!!9J!t~ wi th i nrl uenza A Oontrol 7 Days 9 11 13 15 Percentage of tracheal explants show1ng absence of ciliary activity when infeoted with H. pulmon1s followed by influenza A. 78 of 103 TCIDSO per tube. On day three 10 3 • 4 CFU of M. pulmonis was added to 3 virus infected and 3 mycoplasma control organ culture tubes. The titrations and ciliary observations were conducted in the same manner as described in the previous experiment. Interpretation of the data presented in Table X indicates again that the mycoplasma titer of the supernatant culture fluid does not differ significantly between the dually infected and mycoplasma control cultures. The virus yield was not reduced to the same degree when the mycoplasma was added 3 days after as when it was added 3 days before infection with the virus. By day 15 there was o~ly a 0.7 log' difference in the virus titers of the virus control cultures compared with the dually infected organ cultures. A comparison of the data on the ciliary activity shows that the combined infection was more damaging than the s1ngle infection by the virus. The single virus infection was more damaging than the single infection by the ~. pulmonis. Figure 16 is a graph of the supernate titers for the two organisms and Figure 17 is a graph showing percent inhibition of ciliary activity. The third experiment was deSigned to determine the effect of a dual infection when both organisms are introduced at the same time. On day zero 3 tracheal organ cultures were inoculated with 103 • 3 TCID 50 of influenza A virus and 3 cultures were inoculated with both organisms 79 Table X Influenza A Virus Followed by Mlcoplasm~ in Mouse Traoheal Organ Oulture Test 0 P~W~J1~~ DalS iostl~oulatJ~~~ 11 3 7 9 5 1 ~ 13 15 Mean titer Ma 3.4 5.8 6.0 5.9 Mean titer )lIb 3.4' 5.9 6.5 5.8 5.6 5.7 5.5 2.4 2.; 2.6 2.1 2.4 1.8 1.5 1.4 1.0 1.0 1.0 Mean t1ter VO 3.0 Mean titer Vd ;.0 2.4 2.6 JI-.8 2.0 5 e O 505 2.5 1.7 Oiliary act1vitye 0 0 0 0 0 0 2 10 45 Influenza A 0 alone 0 0 2 11 21 31 44 70 !I. ;rmonlS 0 0 0 23 30 40 55 62 91 Un1n.tected control 0 0 0 0 0 0 0 0 0 M. Eulmonis - alone P us Influenza A a LoglO OFU per ml!. pulmonis cultured alone. b LoglO OJ'U per ml M. pulmonl,s cultured with influenza A. c LoglO T01D50 per ml influenza A oultured alonee - LOS10 TOID~o per ml influenza A cultured lnth M. ;eulmon1 • e 'eroent of--tracheal explants showing no ciliary activity. d 80 11. Rta-lmonl!, OA.lOne ~ Dual 1nfect1on Logl O oru per ml Influenza A o LoglO T01D50 per ml o 1 Figure 16. A.lone ~ Dual 1nfect1on 3 5 7 Days 9 11 13 15 Yield of ~. pulmonis and influenza A virus from supernatant medium of dually infeoted mouse traoheal organ oultures when influenza A virus was inoculated first. Broken line indicates the limit of sensitIvity of the assay system. 81 Percent absent ciliary activity 0 0 H. 6; 6 Influenza A alone 0 CJ Influenza A with M. pulmonis • pulmoP1s alone - -e Oontrol '/. "\ 1· 100 o 1 3 5 7 Days 9 11 13 15 " F1gure 17 • Percentage of tracheal exp1ants showing absence ot oiliary act1vity when infeoted w1th 1nfluenza A/PR-8 tollowed by!. pulmonls. 92 in the above concentrations. The titrations and ciliary ob- servations were conducted in the same manner as described in the previous experiment. Table XI shows that the super- natant titers for the mycoplasma again were not affected the Simultaneous growth of the virus. ~ The virus titers however, dropped off significantly (5% significance level) starting about day 5 as shown by the data in Figure 18. By day 15 the mean virus titer yield in the dually infected culture supernate was only 100.1 compared with a mean titer of 101 • 8 in the singly infected culture. The ciliary activity was inhibited earlier in the cultures with the mixed i~fections than in the cultures with the single infections as shown by the graph in Figure 19. By day 5, 30% of the tracheal explants scored for activity in the dually infected cultures were inhibited whereas none of the explants in the singly infected cultures showed c11iostas1s. It appears then that the dual infection is more destruct.:l.va than the single infections when ciliary activity is used as an index. G. Histopathology The histopathology as revealed by the Hand E stained sections appeared earlier in the combined infection. By the 5th day the epithelial layer was nearly completely destroyed. Figure 20 shows a comparison of the histology of trachea infected with ~. pulmon1s alone and trachea after 83 Table XI Influenza A Virus and MYoo~asma pulmonis in Mouse Tracheal Organ au. ture - Test 0 1 Day! post1nocu~~ 11 9 5 7 3 15 4.8 4.,7 4.:; 4.,2 307 5.9 5.8 5.2 4.8 4.3 4.2 ,.0 Mean titer ~ 4.1 5,1 6.3 Me"an t1ter Mb 4.1 5.6 Mean t1ter VC 3.3 2.6 Mean t1ter Vd :;.:; 3.2 13 4.8 2.8 2.5 2.7 2.7 2.2 2.1 1.8 :;.3 0.8 0.,3 0.1 0 41 2 0.5 0.1 Oiliary actlv1ty e M. 12ulmonis - alone 0 0 0 0 0 2 7 38 67 Influenza A 0 alone 0 0 0 12 27 42 87 94 0 0 30 44 60 84 100 100 0 0 0 0 0 0 !I. E.ulmonls 0 plus Influenza A Uninfected control 0 0 per ml influenza A cultured alone. TOIDSO 0 b LaSlO OFU per ml M. pulmonkJ! cultured alone. LoglO OPU per ml M. pulmon1Jl cultured 'tr1 th influenza: A. 0 L0810 a TC1DSO per ml influenza A oultured with M. ulmonis. e Peroent of tracheal explants showing no ciliary aot1vl ty. d LOg~o 84 11. pu1monis o Alone ~ Dual infection Log lo OPU per ml 6' 5 4 3 Influenza A virus Log lo TCID50 per m1 DAlone ~ Dual infection :5 2 1 o o 1 3' 5 7 9 11 13 15 Days Figure 18. ~ield·of Ii_ pulmonla and influenza A virus from supernatant medium of dually infeoted mouse traoheal organ oultures when both organisms are inooulated at the same time. Broken line indioates the limit of sensitivity of the assay system. 85 Percent absent c1liary aotivity 0 0 !. pulmonis alone 6 6 Influenza A alone 0 D Influenza A w1th M. Rulmon1s • • Control - 100 80 60 40 20 o F1gure 19. 5 7 Days 9 11 13 15 Percentage of trache81 explants showing absence of ciliary activity when infected wi th 1nfluenza A/PR-8 and .H. pulm'onis at the same time. 86 A F1qure 20. B Appearance of mouse tracheal explant after 5 days in culture. (A) Infected with ~. pulmon1s, note cilia (arrow). (B) Infected with ~. pulmonis and influenza A/PR-8. H&E stain X 400. 87 combined infection after 5 days infection. Although some damage is noted in the section from the sinql'e infection, the dual infection had resulted in a complete loss of the epithelial layer in most cases. Figure ~l shows a com- parison of the histopatholoqy of tracheal explants infected with influenza both organisms. A alone and in a dual infection with 'While the tracheal section from the single infection shows considerable disorganization of the epithelium, the combined infection has again resulted in a total loss of the epithelial layer. The tissue represented in these figures was from the experiment involving inoculation of both organisms on day zero. This experiment demonstrated the greatest difference in histopathology on day 5. H. Electron Microscopy An electron-photomicrograph of mouse tracheal ex- plants after 24 hours culture in the presence of influenza A and ~. pulmonis is shown in Figure 22. Many mycoplasma bodies can be seen among the cilia on the surface of the epithelial cell membrane. The mycoplasma are sitting in cup-like depressions between the microvilli. were observed within any of the cells. also be seen attached to the cilia. peared to be clumped together. No mycoplasma Virus particles can The cilia again ap- No evidence of viral cytoplasmic inclusions were seen. Sections observed after 88 A Figure 21. B Appearance of mouse tracheal explant after 5 days in culture. (A) Infected with influenza A/PR-8. (B) Infected with influenza A and M. pulmonis. H&E stain X 400. 89 Figure 22. Eleotron micrograph ot M. pulmonis (M) and influenza ~PR-8 virus Tv) aual infection of mouse traoheal explant atter 1 day 1n culture X 30,000. 90 lonqer incubation periods revealed cell injury demonstrated bf loss of cilia and numerous cytoplasmic vacuoles. DISCOSSION Mouse tracheal organ cultures provide a means of studying the growth propert1es of mycoplasma and viruses in differentiated ti~A\les. The organ culture technique permits one to examine functional changes such as ciliary inhibition as well as cytological changes induced by the infecting organisms. The study of respiratory-tract in- fections with the use of organ cultures has so far been very limited (Hoorn and Tyrrell, 1969). The use of tra- cheal organ cultures in the study of mixed virus-mycoplasma infections has not yet been reported. MYcoplasma pulmonis when cultured alone in mouse tracheal organ culture grew well. Cherry and Taylor- Robinson (1970b) reported that, of 17 strains of mycoplasma studied, all increased in titer when cultured in chicken tracheal organ cultures. It was not stated whether or not the culture medium was completely removed when the titrations were made. They found that seven strains of ~. H. mycoides inhibited the ciliary activity. study M. pulmonis caused progressive damage to qallisepticum and In this the tracheal tissue manifested by ciliary inhibition and cellular degeneration. ~. Collier et ale (l969a) reported that pneumon1ae likewise caused cytopathology in hamster tracheal organ cultures but four other human species of 92 mycoplasma failed to produce any cellular damage. They re- ported that the time required for disappearance of c111a~y activity was inversely related to initial inoculum size. Organick and Lutsky (1968) reported that intense fluor.escence was seen at the bronchial epithelial sur-face when they examined lung sections from N. pulmonis infected mice. Similar fluorescence was observen on the surface of the tracheal epithelium from infected explants in the present study. Small patches of fluorescence in the loose connec- tive tissue of the explants was also observed when the trachea had been infected for 11 days. The fluorescence observed in this study was never seen within the cell, only on the cell surfaces. Collier et ale (1971) stated that the degree of fluorescence seen in fluorescent-antibody atained hamster trachea infected with ~. pneumoniae correlated with the ciliary inhibition. This correlation was also noted in this study, that is the more intense the fluorescence the greater the degree of ciliary inhibition. When the mouse tracheal explants were examined by electron microscopy the mycoplasma were seen to reside in close association with the cell membrane. The M. pulmonis was frequently seen in cup-like depressions in the cell membrane but, no mycoplasma were seen within the cells themselves. Zucker-Franklin et ale (1966) studied the growth of mycoplasma in HeLa cell cultures and also found that the mycoplasma resided on the cell membranes but were 93 never found within the cells unless the cells had become necrotic. Collier at ale (1971a) studied Me pneumoniae infection in hamster tracheal organ cultures by electron microscopy. The results they reported were similar to those reported here. They found the mycoplasma were attached to the cell membrane at the base of the cilia and in the intercellular spaces between the cells. They also failed to find any organisms within the cells. ~. Eulmonis induced cytopathic effects in BeLa cells by its intracellular multiplication reported Hummeler and Armstrong (1967). The mycoplasma were never found extra- cellularly they reported. Edwards and Fogh (1959) also reported observations by electron microscopy of mycoplasmas within cells. The organisms were seen both on the cell surface and within the cytoplasm of FL cells. The intra- cellular presence of the mycoplasma was always associated with cell necrosis they stated. Anderson and ~anaker (1966) found that mycoplasma strain 880 grew on the surface and within mouse fibroblasts (L-cells) however, Jones and Hirsch (1970) found that M. pulmonis grew on the surface of L-cells but never invaded the cells. These contradic- tory findings indicate the confusion that exists regarding the location mycoplasma when grown in cell culture. Collier and Clyde (1971) reported finding a distinctive morphological structure of tl. pneumoniae which appeared to play a role in attachment of the organism to the ciliated 94 cell membrane of hamster trachea. seen with the M. No such structures were pulmonis under electron microscopy re- ported in this study. Results of the study of the effect of receptor destroying enzyme eRDE) on the growth and ciliary inhibiting effect associated with the culture of M. Eulmonis in tracheal organ culture indicated that RDE did not alter the growth characteristics of this organism in the culture system. Manchee and Taylor-Robinson (1969a) reported that colonies of H. pulmonis readily adsorbed fowl, human erythrocytes, and HeLa cells but that sialic acid receptors were probably not involved since treatment of the cells with RDE or swine influenza virus failed to inhibit the reaction. The un- altered growth characteristics noted in the present study seems reasonable since the RDE most likely did not affect the mycoplasma-cell association. Gesner and Thomas (1965) suggested that N-acetyl neuraminic acid (sialic acid) containing receptor sites on turkey erythrocytes were responsible for the agglutination of these cells by M. gallisepticum. t1anchee and Taylor- RobinSOn (1969b) in another study reported that sialiC acid at the surface of HeLa cells was involved in the adsorption of these cells:.to colonies of also that colonies of adsorbed to HeLa mechanism since ~. !:!~ eneumoniae. They found hominis and M. salivarium also cells, but apparently by a different RDE failed to inhibit adsorption of the cells 95 to these colonies. It thus appears that M. pulmonis organ- isms do attach to the mouse tracheal epithelial cells but sialic acid receptors are not involved. ~. The growth of pulmonis was also shown to require viable tracheal tissue since culturing of the organisms in cultures of heat growth. t.r.e~ted tracheal tissue resulted in no It was previously established that the L-15 growth medium alone would not support the growth of this mycoplasma. An experiment was undertaken to determine what might be added to the cultures of heat treated tissue to enable the mycoplasma to grow. It was found that the addi- tion of cholesterol or 5% horse serum would enable the mycoplasma to grow. growth. The horse serum promoted the best This was not surprising since Razin and Tully (1970) have shown that M. pulmonis requires cholesterol for growth • . The L-1S culture medium, does not contain any sterols. Horse serum, besides providing a source of cholesterol, also contains other lipoproteins known to stimulate the growth of mycoplasmas. It might be concluded that the H. pulmonis obtains its required sterol and possibly other growth factors from the tracheal tissue cells. Cell-free filtrates from ~. pulmonis infected tracheal organ cultures when added to sterile tracheal organ cultures did not produce the ciliary inhibition that was noted in the infected cultures. This indicated that stable toxic metaboliC products which might have caused the ciliostasis 96 were not produced in sufficient concentrations to cause this effect. This could also mean that the ciliostasis was not caused ~ the mycoplasma depleting the modium of some nutrient required by the tracheal ciliated cells. ~. Thomas (1963) demonstrated lethal toxic properties of pulmonis in mice. He [cund that ~. pulmanis was only toxic for rats and mice and that viable orqanisms were necessary to produce the lethal effect. the toxin involved. He did not characterize Cherry and Taylor-Robinson (J.970) conducted a similar experiment trying to determine tho mechanism by which ~. mvcoides produced ciliary inhibition in chick embryo tracheal organ cultures. They also failed to transfer the ciliostatic effect using culture filtrates from infected cultures. They were forced to conclude their experiment after only 7 days because their filtered medium was found to contain viable mycoplasma. effect produced by the ~. The ciliostatic pulmonis demonstrated in the present study could possibly be a result of the production of an unstable toxic substance such as hydrogen peroxide. The production of hydrogen peroxide in close proximity to the cell membrane could possibly cause inhibition of ciliary activity. An experiment was conducted to demonstrate hydrogen peroxide production by ~. pulmonis. When colonies of ~. pulmonis were overlaid with guinea pig erythrocytes the cells adsorbed to the colonies and stained dark blue when 97 the methylene blue stain was added. Sobeslavsky and Chanock (1968) demonstrated hydrogen peroxide production by human and several animal species of mycoplasma by laying growing colonies with guinea pig blood-agar and reading for hemolysis. pulmonis, ove~­ m1xtu~e Of 11 species tested only Me ~. pneumol?:D\:,{~ ~. gallt~cEticum and !:!. neU~Oty.­ ticum produced measurable zones of hemolysis at 24 hou~o, however, all the species tested produced some degree of hemolysis after 96 hours. The hemolysis produced in all cases could be blocked by the addition of catalase. Cole at ale (1968) also studied peroxide production by mycoplasma species and compared several different test systems. also established that M. They pulmonis was a potent hydrogen peroxide producer. Once it was shown that ~. pulmon1s produced pero- xide, an experiment was conducted to determine if the peroxide produced could be responsible for the ciliary inhibition in the tracheal cultures. It has been shown (Cherry and Taylor-Robinson, 1970b) that hydrogen peroxide will inhibit ciliary activity in tracheal organ culture. It was found that the addition of catalase to the medium delayed the initiation of ciliary inhibition hOtlever, it did not completely prevent it. (19700) found that ~. Cherry and Taylor-Robinson pulmonis only produced one-third as much hydrogen peroxide as M. tracheal organ cultures. They found that the addition of rnYco1des !!£. capri in chicken 98 catalase delayed the damage to the tissue caused by eycoides !!£. capri. ~. The addition of exogenous hydrogen peroxide to the tracheal cultures also resulted in the loss of ciliary activity. with H. pulmonis. They did not mention a similar test Brennan and Feinstein (1969) studied the relationship of hydrogen peroxide production b¥ H. pulmonis to its virulence for catalase deficient mice. After infecting mice with the mycoplasma, they found significantly more acatalat1c mice had pneumonia did normal mice. ~ day 3 than They also found that exogenously supplied H. catalase stimulated the growth of longed its survival at 25 C. pulmonis and pro- Their findings suggested to them that hydrogen peroxide production contributed significantly to the virulence of the pulmonis. ~. They also commented that the absence of catalase caused earlier death of the organisms. Brennan and Feinstein (1969) reported that 1010 CFU of M. pulmonis produced 0.065 micro moles of hydrogen peroxide per hour. They also found that ~ adding glucose to a final concentration of 0.01 M the hydrogen peroxide production was increased ~ 50%. In the study reported here glucose was added to tracheal organ cultures in a concentration of 1% and the cultures were then infected with pulmonis. H. It was felt that this might increase with ciliostatic effect produced by the ~. pulmonis if hydrogen peroxide were responsible, for the eiliostasis. The results, 99 however, revealed very little difference in the temporal ... relationship of the ciliary inhibition in cultures containing glucose compared with cultures without glucose. A 50% increase in hydrogen peroxide production if it did occur, may not be enough of an increase to produce a detectable difference in the ciliary activity. If higher concentrations of organisms had been employed an increase in hydrogen peroxide might have produced more obvious effects. Cohen and Somerson (1967) found that M. pneumon1ae also produced more hydrogen peroxide when glucose was included 1n the medium. When glucose was omitted from the medium no hydrogen peroxide was observed. They established that the peroxide was produced directly by the organisms and was not derived indirectly by the aerobic oxidation of some other secreted, autoxidizable substance. Cohen and Somerson (1969) also reported that added glucose stimulated a peroxidase-like activity which rapidly degrad.ed any hydrogen peroxide released in the medium by H. eneumoniae or that ~. ~. gallisept1cum. Low (1971) also found pneurnoniae in the presence of glucose removed both endogenous and exogenous hydrogen peroxide. He also sug- gested that this was due to an inducible peroxidase. Whether or not ~. pulmonis 1n the presence of glucose pro- duces a peroxidase has not been reported. In this study M. pulmonis was also cultured in tracheal organ cultures in the presence of amino-tr1azole. This 100 was done since Brennan and Feinstein (1969) reported that amin-triazole effectively inhibits catalase that may be produced endogenously 1n the cultures by tissues. catalase were be1ng produced ~ If the explants and this was partially degrading the peroxide produced by the mycoplasma then the addition of amino-triazole should enhance the ciliary inhibitory effect produced by the by binding endogenous catalase. ~. pulmonis The addition of the amino- triazole, however, did not produce any significant difference in the ciliostatic effect produced by the mycoplasma. This must mean that catalase was not being produced by the explants in any siqnificant amount or that the test system was not sensitive enough to detect the difference. These studies indicate that ~. pulrnonis caused a toxic effect in mouse tracheal orqan cultures. This effect was delayed by the addition of catalase to the medium. These observations plus the fact that the organisms were observed to be closely associated with the cell membranes of the tracheal tissues may account for the c1liostasis and histopathology. Toxic actions of peroxide on various enzyme systems and tissues have been well established (Cohen and Somerson, 1967). virulence of ~. Cohen and Somerson (1967) felt that the pneumoniae was due to the hydrogen peroxide secreted b¥ the organisms growing on the tissues of the respiratory tract. The work of Lipman et ale (1969) indicated that the pathog,enicity of ~. pneumoniae was related to the organisms' ability to hemadsorb sinco strains of M. pneumoniae which had lost their hemadsorpt1on capacity were found to be non-pathogenic. reported ~ In another study Lipman and Clyde (1969) they concluded that the possession of intense hemadsorbting ability was not a quality unique to v~rulcnt strainn of H. pneumoniaf.!. They also noted that quantitative differences in hydr.ogen peroxide formation was not related to the degree of virulence of ~. pneumoniae. They concluded that cytoadsorption and hydrogen peroxide formation were not entirely responsible for the pathogenesis of this organism. (1971) concluded from their studies of Collier et ale M. pneumoniae :t.nfec- tions in hamster tracheal organ culture, that the close relationship of this organism and the epithelial cell membrane could have allowed either mechanical or chemical injury to occur. They suggested that the masses of organi- sms laying close to the cilia could interfere physically with the normal beating of the cilia or injury of the membrane could have resulted in disturbance of surface charges needed for synchrony of ciliary motion. A similar conclusion could be drawn regarding the observations reported in this study. The b1 electron microscopy to M. pu1monis were also observed be close to cilia which may have physically affected the ciliary activity. The mycoplasma were not seen actually attaching to the Cilia, however. The results reporte~ in this study demonstrated that 102 the influenza A PR-8 virus grew in the mouse tracheal organ culture system through the 15 day test period. When the medium is completely removed for titration and fresh medium replaced the continuous presence of virus in the medium indicates growth (Hoorn and Tyrrell, 1969). Bang and Niven (1958) found that chick adapted influenza virus would not grow in human embryonic thacheal cultures but would however, grow in adult ferret mucosa destroying the ciliated and mucus secreting cells. Harford and Hamlin (1952) found that even though maximal destructive action of influenza A PR-8 virus was found in mouse bronchi on day 2 after infection, the ciliary beat was just as active as in the control mice. Hoorn and Tyrrell (1966) reported that influenza A virus grew in 20 out of 20 human embryo tracheal cultures. The extent of growth of the virus in this system was not reported. Reed (1969) found that an inoculum of swine influenza virus of 101.S TeDSO per ml yielded 10 6 TCDSO per ml of virus after 15 days in calf tracheal organ cultures. However, influenza A (WS) decreased from 102 to 101 • 8 TCD 50 per ml after 15 days in the same culture system they reported. A decrease from 102 to 101 • 8 TCD SO per ml does not appear to be significant considering the assay system employed. Observations of the ciliary activity of the mouse tracheal explants infected with influenza virus in this study revealed reduced ciliary activity starting on day 3 103 with complete ciliostasis commencing on 7. By day 15 from 90-100% of the explants showed complete ciliostasis. Campbell et ale (1969) infected calf tracheal organ cultures with bovine parainfluenza 3 and noted ciliostasis starting at day 5. The effect on the ciliary activity was found to be dose dependent with larger doses of virus causing ciliary inactivation earlier. Hoorn and Tyrrell (1965) reported that influenza A2 virus did not grow well or produce any effect on the ciliary activity in monkey tracheal organ cultures. The same virus did however grow and inhibit ciliary activity in nasal ciliated epithelium of the monkey. When Tyrrell et ale (1965) infected human embryo tracheal organ cultures with 104 eg9 infectious doses (ElDSO) of influenza A2 virus, reduced ciliary activity was first noted on day 5 with complete ciliary inhibition occurring on day 7. The virus titer reached a peak of 105 EIDSO on day 5 then slowly declined to a titer of 102 ElDSO at the end of the experiment on day 15. Herbst-Laier (1969) compared the growth of selected myxoviruses in tracheal organ cultures prepared from tracheal tissues from different animals. He found that influenza A PR-8 virus would grow in tracheal cultures prepared from the tracheas of human embryos, laboratory mice, hamsters, rats, ferrets, dogs, and vervet monkeys. He suggested that good growth of myxoviruses in dog tracheal cultures might suggest a potential reservoir for influenza viruses in these 104 animals. The low susceptibility of pig tracheal explants to human influenza viruses could be due to a latent mycoplasma infection in the respiratory tissue of these animals, he commented. In the present study histologic examination of the explants from the virus infected cultures revealed progressing damage to the ciliated epithelium resulting in complete denuding of the epithelial surface of most of the explants ~ day 11 to day 15. Hers (1966) stated that since the growth cycle of influenza virus is short, i.e., about 8 hours, the death and resulting desquamation of the affected cells occurs very early in the disease. He pointed out that viral multiplication was the major cause of cell death. The histopathology observed in the studies reported here was very similar to that observed (1969). ~ Reed She studied the action of parainfluenza virus in calf tracheal organ cultures and observed severe damage tO,the superficial layers of the epithelium. Cell debris and vacuoles were seen resulting eventually in complete desquamation of the epithelial layer. She commented that even after the cells were rapidly destroyed virus production continued. It does not seem possible that virus pro- duction could continue after the cells were all destroyed. The electron microscope studies of the virus infected tracheal explants reported here, revealed numerous virus , particles attached to cilia. The virus appeared to cause 105 the cilia to clump together probably due to the attached virions forming bridges between the cilia. Viral inclu- sions were not seen in the cytoplasm of the tracheal epithelial cells. Virus particles were seen close to and on the cell membrane but virus was not seen undergoing egestion from the cell. Loosli et ale (1970) in their electron microscopic examination of influenza A PR-B virus in infected mice reported observing virus particles being shed from the surface of secretary Clara and ciliated cells at 12 and 24 hours after first exposure of the mice to the virus. They also reported observing viral cytoplasmic inclusions and marked destruction of the ciliated cells b¥ 72· hours. Harford et ale (19~5) infected mice by aerosols of influ- enza A PR-S and studied the infected tissue by electron microscopy. They also reported finding cytoplasmiC in- clusions which occurred in areas of the cytoplasm well separated from nUCleus, cell membranes, endoplasmic reticulum, mitochondria or lipid granules. The inclusions that they observed contained virus particles. They pointed out ,that fluorescent antibody studies by others had not revealed isolated areas of cytoplasmiC fluorescence which might correspond to the viral inclusions that they observed. The experiment reported in this study concerning the effect of receptor destroying enzyme (RDE) on the growth of 106 influenza A virus in the mouse tracheal organ culture yielded interesting results. It has been shown by Marcus et ale (1965) and many others that myxoviruses specifically attach to terminal neuraminic acid groups on qlycoproteins of the cell membranes and that myxovirus attachment can be prevented ~ the prior treatment of the cells with RDE. The fact that the virus growth was reduced rather than completely blocked in the present study probably indicates that a greater concentration of RDE might have been more effective in preventing the infection. The fact that the virus titer appeared to increase again after day 7 might be explained b¥ the observation reported by Marcus and Schwartz (1968). They found that after the sialic acid receptors have been once destroyed ~ the RDE, new recep- tors appear on the cell membrane within 8 hours. The increased growth later in the test period might have been prevented ~ continuously adding RDE to the cultures throughout the experimental period. When the influenza A PR-8 virus was cultured in a dual infection with ~. pulmonis in the mouse tracheal organ cul- ture, reported in this study, the ciliary activity was inhibited earlier than when either organism was cultured alone. The dual infection also resulted in earlier his- topathological changes. This enhanced pathology might be described as evidence of synergism employing the broad definition of synergism used by Lepper (1968), i.e., a more 107 sever disease produced ~ either agent alone. ~ the dual infection than produced This might be considered additive according to ones definition of synergism. Ranck et ale (1970) reported a synergistic action when they injected both influenza A virus and ticum together into turkey poults. ~. gallisep- The combination pro- duced more extensive lesions in the air sacs than was produced by either agent alone. Heishman et ale (1969) also reported a synergistic action between a virus and a mycoplasma resulting 1n production of a more severe airsacculitis 1n chickens. combination of ~. The mixed infection was produced by a gal11sept1cum and either Newcastle disease virus or chicken infectious bronchitis virus. The growth rate of the mycoplasma appeared not to be affected ~ the simultaneous growth of the virus however, the viral replication was inhibited b¥ the growth of the mycoplasma. The results of Nakamura and Sakamoto (1969) indicated that influenza A virus was inhibited by the simultaneous growth of ~. eneumoniae in cell culture. They found that the in- hib1tion could be reversed by the addition of arginine to the culture medium. This is hard to understand since H. pneumoniae does not dissimulate arginine like most other mycoplasma. Rouse et ale (1963) reported a lowered plating efficiency of adenovirus 1n KB cells contaminated with mycoplasma. They felt that this inhibition was due to the mycoplasma depleting the growth medium of arginine, an 108 absolute requirement for growth of the tissue cells. When arginine is metabolised by mycoplasma, ammonia is produced (Schimke and Barile, 1963). Jensen et ale (1961) demonstrated the inhibitory action of ammonium ions on the growth of influenza virus. a mechanism for the virus by the M. ~T,nwth This is unlikely to be inhibltton of the influenza pulmonis reported in this study since ~. pulmonis does not utilize arginine as its energy source. Butler and Leach (1964) attributed the decreased growth rate of measles virus grown in HEp-2 cells in their laboratory to the presence of an acid-producing mycoplasma. The H. pulmonis used in this study is known to produce acid from glucose, however, there is no glucose in the L-15 medium employed in this study therefore, growth inhibition of the influenza virus due to acid conditions is unlikely. The pH of the medium in this study was well maintained by complete medium changes every 48 hours. Powelson (1961) studied the effect of mycoplasma on the metabolism of tissue cultures. Her studies indicated that m¥coplasma considerably alter the amino acid metabolism of mouse fibroblasts. Viral replication requires a complete functional amino acid pool in the host cell, thus' if the H. pulmonis significantly alters metabolism of the epithelial cells in the mouse organ culture then inhibition of influenza A virus growth might be expected. MYcoplasma have also been shown to alter nucleic acid 109 metabolism of mammalian cells. reported that H. stock and Gentry (1969) hominis contributed significant deoxyribo- nuclease activity to virus infected as well as virus free cell cultures. Randall et al. (1965) also noted that HeLa-cell cultures infected with mycoplasma demonstrated very unstable (i.e., rapidly degraded) host cell DNA. They also reported that DNA from mouse derived L-cells infected with mycoplasma was very unstable. Mycoplasmal DNase activity could conceivable degrade a replicating viral genome of a DNA virus but would probably not affect the genome of a RNA virus such as influenza A. Nardone et ale (1965) found the mycoplasma completely inhibited the incorporation of tritiated thymidine and uridine by L cell cultures. This inhibition of incorpora- tion was a result of inhibition of nucleoside uptake mrcoplasma infected cells. ~ This could affect the growth of a virus infecting the same cells since viral replication requires a pool of phosphorylated nucleosides. One other mechanism that must be considered as a possible mechanism for the reduced growth of the influenza virus in the dually infected tracheal cultures is the possible induction of interferon production , by ~. pulmonis. ;. Tracheal organ cultures have been known to produce significant levels of interferon. When Willems and Van der Veen (1968) infected mouse tracheal organ cultures with Sendai virus tests for interferon were positive at 2 days. 110 Interferon was produced equally well at either 33 C or 35 C of incubation. Reed (1969) reported interferon pro- duction in pig tracheal organ cultures infected with swine influenza virus, however, treatment of pig tracheal cultures with interferon or specific antibody did not prevent infection of the organ cultures. Interferon has also been produced in calf tracheal organ cultures. Smorodintsev (1968) found that Sendai virus could elicit interferon titers as high as 1024 units within 3 days when 10 5 TC1DSO of virus was inoculated into the cultures. The titer had dropped to zero by the 5th day however. It is still an open question as to whether or not mycoplasma can induce interferon production. Armstrong and Paucker (1966) studied the effect of mycoplasma on interferon production in L-cells and human embryonic kidney cells. Of four species of mycoplasma they studied, none produced detectable levels of interferon. They also noted that the responsiveness of the cells to induction of interferon by virus remained unaltered. The infection of the cells with mycoplasma did not impair the sensitivity of the cells to the action of interferon either, they reported. A separate study by Yershov and Zhdanov (1965) also indicated that mycoplasma would not induce interferon production. However, they found that pretreatment of their primary chick embryo fibroblast cultures with mycoplasma before virus infection increased the production of interferon 111 about 4 times, after the virus was introduced suggesting a priming effect. Whether or not interferon played a role in the reduced growth of the influenza virus noted in this work was not studied. This study is believed to be the first concerning virus-mycoplasma interaction in motlSe tracheal organ culture. A synergistic action was demonstrated in this experimental model. This suggests the possibility of virus-mycoplasma interactions occur.r.ing in respiratory infections of man. SUMMARY It was found that the ~. pulmonis grew well in the mouse tracheal organ culture system producing inhibition of the ciliary activity. The orqanism grew in close prox- imity to the tracheal epithelial cell membranes producing cytopathology as revealed by stained histological preparations, fluorescent antibody studies, and electron microscopy. The~. pulmonis would not grow in the organ culture growth medium alone but grew when viable tracheal explants were present. Cultures of heat treated explants with added cholesterol or horse serum supported growth of the mycoplasma. The~. hydrogen peroxide. pulmonis was shown to produce The addition of catalase to the mouse tracheal organ cultures infected with this organism appeared to protect the explants from the toxic effects produced by the mycoplasma as indicated by inhibition of ciliary inactivation. There was no ciliary inhibition in cultures that were maintained in medium in which ~. Eulmonis had been grown but was removed b¥ filtration and treatment with antibiotics. This indicated that the organisms must be present to produce ciliary inhibition and that stable toxic products produced ~ the organism were probably not responsible for this effect. 113 Influenza A/PR-8 virus was also shown to replicate and produce cytopathology in the tracheal organ cultures. Electron microscopy showed that the virions attached to and caused clumping of the cilia. The virus infection eventually resulted in complete desquamation of the epithelial surface of the tracheal explant as revealed by stained histological sections. The addition of receptor destroying enzyme (RDE) to the medium was found to significantly inhibit the growth of the virus in this system. When tracheal organ cultures were dually infected with both organisms, ciliary inactivation and tissue damage occurred earlier than when the organisms were cultured separately. 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