‘ . 4 a” . Dr, . ‘II . ‘0. . . fle‘V egre- _ HIT '."-.',Ni STATE“ U'NNi ..‘Cvi;.‘. ..‘¢...‘.. T basis for the 'D itfiwk \ ' . x’ .. _. . I)». ...g,n.a...,..,.( ,m...«&4; 1“ a w .. 5.7. RSITY J m C E ‘ . . n... ... q . . _ 1.. .. . ‘r . . . C . . ‘ » y - .. . . a . . .. q .. V _. . - . _ L .. . ‘ ., . . ~ . . . i .: . . .. x . . . 1 yr; .H. .4 . .. . Linn .. ‘ .‘ -, . 1 . ?‘ k .. 2-.. . C .82... .74. :HM. 0". M . I '0 1‘ n -. xvi.» I. c I. o 1.. _‘A.- .' 4i v..l1" . ‘1. I: nIu.V - '4 1'1. Ill I I‘V‘ " A I: “Ccanci gixlsnggyvéy :A.$r.h~..«.c\r5.. 51-, w I... :2. C» i. . 4... - I: .. , v ' Er ‘L- l l ' I I' ' ‘ I- ' ' o _ I — I - -_ l rm“ r. 1'.” Michigan State University This is to certify that the thesis entitled THE PATHOLOGY OF MYCOPLASMA PULMONIS INFECTION IN THE RAT presented by GEORGE C . JERSEY has been accepted towards fulfillment of the requirements for Ph . D . . PATHOLOGY degree in Major professor Date May 16, 1973 0-7 639 - 3.93m: av 7" HOME 8: SUNS’ BOOK BINDERY INC. LIBRARY BINDERS Quantitflfig! A ABSTRACT THE PATHOLOGY OF MYCOPLASMA PULMONIS INFECTION IN THE RAT BY George C. Jersey A total of 142 germfree and ex-germfree rats were used in 11 trials to reproduce experimentally chronic respiratory disease (CRD) of rats, and to isolate and identify the etiologic agent. The first 3 trials were designed to evalu- ate natural transmission of CRD from diseased rats, that had been collected from 5 different sources, to ex-germfree rats. These experiments were followed by 8 trials using germfree and ex-germfree rats to experimentally reproduce CRD, using either a cell-free filtrate prepared from pneumonia lungs or broth cultures of Mycoplasma pulmonis. Following exposure to the filtrate or culture, the rats were killed at selected intervals and examined for microbial and morphologic evidence of CRD. Respiratory lesions typical of the natural disease developed in adult rats within 2 to 6 weeks following inocu- lation. Young rats were resistant to infection and developed lesions only after prolonged incubation, if at all. In one experiment, young rats were maintained for 7 months after George C. Jersey inoculation without developing pulmonary lesions. The clinical signs in experimentally infected rats were rough hair coat, dyspnea, anorexia, loss of weight, and humped posture. All principal clinical signs and gross and microscopic lesions of CRD were reproduced in 32 of 54 adult germfree and ex-germfree rats. Gross lesions were pneumonia characterized by red and gray areas of consolidation and bronchiectatic bronchi containing muc0purulent or caseopurulent exudate. Microscopic lesions included bronchiectasis, bronchial epi- thelial hyperplasia and necrosis, mucopurulent bronchial exudation, peribronchial and perivascular lymphoid hyper- plasia, and pulmonary atelectasis. Additional lesions were tracheitis, otitis media, and rhinitis. Exceplasma pulmonis was re-isolated from 40 of 65 lungs, 13 of 13 tympanic cavities, and 8 of 8 tracheas of germfree or ex-germfree rats following inoculation with aerosols of cell-free filtrate or broth cultures of Mycoplasma pulmonis. The organism was identified by a growth inhibition test. It is concluded that flchpIasma pulmonis is the primary cause of CRD in rats and the appropriate name for the disease is respiratory mycoplasmosis. THE PATHOLOGY OF MYCOPLASMA PULMONIS INFECTION IN THE RAT BY George C? Jersey A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pathology 1973 ‘15..“ Dedicated to Barb, Cindy and Kay ii ACKNOWLEDGEMENTS The author wishes to express his appreciation and gratitude to the following: To Dr. C. K. Whitehair, my major professor, for his assistance in providing funds and resources and his patient counsel and guidance in the conduct of this research. To Dr. G. R. Carter for providing laboratory facili- ties and his guidance on microbiologic methods and techni— ques. I am especially indebted to Mr. A. Wayne Roberts, Mrs. Marigrace Shue, and Mr. Harold A. McAllister for their faithful and unceasing advice and assistance in the micro- biologic procedures. To the other members of my guidance committee, Drs. R. F. Langham, R. L. Michel, and O. Mickelsen, for their suggestions and guidance in conducting this research. To Dr. C. C. Morrill, Chairman, and the faculty and staff of the Department of Pathology for providing the facilities, technical assistance and the opportunity to pursue graduate study. To Mr. Larry Spencer and Mr. James Southern for their reliable care and maintenance of the germfree rats which was an essential part of this research. iii To my wife, Barbara, for faithful support and encourage- ment, and for her help during the preparation of this manu- script. This research was supported in part by Grant No. FROO38- 03 from the National Institute of Health, Bethesda, Maryland and by a General Research Support Grant from the College of Veterinary Medicine, Michigan State University. iv TABLE OF CONTENTS Page INTRODUCTION .......................................... 1 LITERATURE REVIEW ............... . .................... . 5 Early Reports.......... .............. ............ 5 Etiology of CRD... ....... . ................ 9 Current Status ....... .. ...... ............... 9 Mycoplasmas....... ................ ..... ..... 11 Viruses........... ..... .. ..... .............. 25 Bacteria.................................... 29 Clinical Signs ..................... . ...... 33 Pathology................. ..... . .......... 34 Respiratory Disease Control...................... 40 Summary of Literature Review..................... 42 OBJECTIVES.......... ..... ..... ........... ...... ....... 43 MATERIALS AND METHODS. ..... ........................... 44 Experimental Rats................................ 44 CRD-Rats.................................... 44 Germfree Rats............................... 45 Ex-germfree Rats............................ 45 Experiments...................................... 46 Exposure to Natural Disease................. 45 Experiment l........................... 47 Page Experiment 2 ............ ....... ..... ........ 47 Experiment 3 ..... .............. ............ . 47 Exposure by Aerosol Inoculation,,,,,......,.,,.,. 48 Experiment 4 .............. ...... ............ 48 Experiment 5 ................... ............. 49 Experiment 6..... ..... ................. ..... 49 Experiment 7. ..... ...... ......... .. ........ . 50 Experiment 8 ................. ... ............ 50 Experiment 9.. .......... .. ..... ............. 50 Experiment 10... ............ .. ..... ......... 51 Experiment 11. ......... . ............ ........ 52 Colony Controls ........................... .. 52 RationS.......... ..... . ...... . ................... ..... 52 Germfree TechniqueS.... ..... ...... ......... . .......... 54 Preparation of Cell-Free Filtrate.............. ....... 55 Techniques for Isolation and Growth of M;_Pulmonis.... 55 Media... ..... ........... ..... .................... 56 Isolation of M; pulmonis from lung............... 57 Isolation of M;_pulmonis from trachea............ 57 Isolation of ML pulmonis from tympanic cavity.... 58 Identification Of M_‘ Enlmonisoooooooooooooocoo. 0000000 58 Preparation of M; pulmonis for Inoculation....... ..... S9 Passage of Infectious Inocula into Isolators.. ...... .. 60 Aerosol Inoculation of Rats........................... 61 Gross and Microscopic Examination of Tissues.......... 62 vi Page RESULTS...... ........ .................................... 64 Experimental Infections.............................. 64 Exposure to Natural Disease..................... 64 Experiment 1........... ........ ...... ....... 65 Experiment 2 .................... ... ..... .... 65 Experiment 3.................... ....... ..... 68 Exposure by Aerosol Inoculation...... ...... ..... 69 Experiment 4................................ 69 Experiment 5 ........ ........... ......... .... 72 Experiment 6.. ....... ... ...... ....... ....... 72 Experiment 7............... ...... ..... ...... 73 Experiment 8.... ..... .. ............. ........ 73 Experiment 9..................... ....... .... 74 Experiment 10 ........ ............... ........ 7S Experiment 11........... ...... ....... ....... 75 Clinical Signs.. ......... . ..... ................. 78 Pathology....................................... 80 Gross Pathology ..................... . ....... 81 Histopathology. ...... .... ......... ..... ..... 8S Microbiology. ....... . ....... ... ......... .... 103 DISCUSSION............................................... 110 Exposure of Ex-Germfree Rats to Natural Disease. ..... llO Aerosol Inoculation Experiments...................... 111 Pathology and Etiology............................... 114 vii Page RESULTS........ ....... ................................... 64 Experimental Infections........................ ...... 64 Exposure to Natural Disease ...... ............... 64 Experiment l........... ...... .... .......... . 65 Experiment 2 ............ . ..... .............. 65 Experiment 3................................ 68 Exposure by Aerosol Inoculation.... ............. 69 Experiment 4 ..... ........................... 69 Experiment 5 .......... . ....... .. .......... .. 72 Experiment 6 ......... ............. ....... ... 72 Experiment 7 ....... ...... .......... ......... 73 Experiment 8 .......... .. ............ ........ 73 Experiment 9......................... ....... 74 Experiment 10............................... 75 Experiment 11.. ............... ... .......... . 75 Clinical Signs ........... . ..... . ...... .......... 78 Pathology........ ........ . ........... . ......... . 80 Gross Pathology ................... . ......... 81 Histopathology ........ . ............... . ..... 85 Microbiology... ............. . ............... 103 DISCUSSION ....... ........................................ 110 Exposure of Ex-Germfree Rats to Natural Disease...... 110 Aerosol Inoculation Experiments...................... lll Pathology and Etiology............................... 114 vii Page SUMMARY... ........... .................................... 120 REFERENCES ............................................... 122 VITA... .................................................. 131 viii Table LIST OF TABLES Page Summary of experimental design used in aerosol exposure experiments................. 53 Summary of data from exposure of 52 ex-germfree rats to conventional rats affected with naturally acquired CRD.. ....... 66 Summary of data from aerosol inoculation of germfree and ex-germfree rats with cell- free filtrate of pneumonic rat lungs or broth cultures of Mycoplasma pulmonis........ 7O ix LIST OF FIGURES Figure Page 1 Vacuolar macrophages and amorphous debris in alveoli of lB-months-old germfree rat..... 77 2 Higher magnification of an area from Figure 1.0.0.00000000000..........OOOOOOOOOOOOIOOOOQ 77 3 Healthy adult male germfree rat......... ..... 79 4 Adult male rat three weeks following inoculation with aerosol of broth culture OfPL{EnlmoniSOOOOOOO......OOOOOOOOOOOOOOOOOO 79 5 Hilar view of individual lobes of lungs from germfree rat in Figure 3................ 82 6 Pneumonic lungs of rat four weeks follow- ing aerosol inoculation with M;_pulmonis..... 82 7 Dorsal view of lungs 15 days following aerosol inoculation with M;_pulmonis... ...... 84 8 Ventral view of lungs 20 days following aerosol inoculation with M;_pulmonis......... 84 9 Sections through cardiac and diaphragmatic lobes of right lung and left lung from an lB-months-old germfree rat................... 87 10 Sections of lungs from rat 17 days follow- ing aerosol inoculation with M;_pulmonis..... 87 ll Transverse sections through totally consoli- dated left lung of rat 16 days following aerosol inoculation with M; pulmonis......... 88 12 Bronchiectasis and atelectasis in lung of rat 35 days following inoculation with M; Pulmonj-SOOOOOOOOOOO............OOOOOOOOOOO... 88 13 Pulmonary consolidation with prominent atelectasis and bronchiectasis from rat 15 days following aerosol inoculation with M; pulmonis..................................... 89 Figure 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Page Division of bronchi with adjacent alveolar tissue in lung from germfree rat............. 89 Acute purulent pulmonary consolidation in rat 16 days following aerosol inoculation Withr2:-EUlmonisooooooooooc00.00.00000000.... 90 Higher magnification of a portion of Figure 15.00000... 000000 0.0.00000.000.000 00000 0.0000 90 Bronchial epithelial hyperplasia and ate- lectasis in lung of rat 16 days following aerosol inoculation with M;_pulmonis......... 92 Hyperplastic bronchial epithelium lining bronchus of rat 18 days following aerosol inoculation with M; pulmonis................. 92 Lymphoid tissue in wall of bronchus from adult germfree rat........................... 93 Lymphoid aggregate in wall of bronchus from adult germfree rat........................... 93 Peribronchial lymphoid hyperplasia in rat 16 days following aerosol inoculation with M; pulmonis.................................. 94 Exudate-filled bronchus from rat 18 days following aerosol inoculation with M; EUlmonj-SO0000000.000.000.00000.00000.0.000000 94 Bronchiectasis and partial atelectasis in rat 18 days following aerosol inoculation Withg:-EUImoniSO00...000000000.000000000.0.0 96 Intrabronchial abscessation in rat 19 days following inoculation with aerosol inocu- lation with M;_pulmonis...................... 96 Small bronchi in lung of adult germfree rat0000000.000.0000.000.00.000.000.0.0.0000.0 97 Distortion of small bronchi ensnared by maturing collagen fibers..................... 97 Transverse section of trachea from rat 15 days following aerosol inoculation with M; enlmonis000000......000.00000000000000000000. 98 xi Figure 28 29 30 31 32 33 34 35 36 37 38 39 4O 41 42 Page Transverse section of trachea from adult germfree rat......... ............... ......... 98 Higher magnification of Figure 27............ 99 Transverse section of trachea from rat 18 days following aerosol inoculation with ML. enlmonis000000000000000.0000.000.000.000.000. 99 Transverse section slightly rostral to external auditory meatus of adult germfree rat00000000.0000000000000000000000000.0000000 101 Transverse section slightly rostral to external auditory meatus of rat 20 days following aerosol inoculation with M. pulmonis...........................TT........ 101 Wall of tympanic cavity from adult germfree rat0000000000000000000000.0000000000000000000 102 Ventrolateral region of tympanic cavity of rat 31 days following aerosol inoculation WithM_o_Bu1m0n150000000000000oooooooooooooooo 102 Higher magnification of area in Figure 34.... 104 Outward extension of otitis media through tympanic membrane of same ear as Figures 34 and 350000.00.00.00000000000.000.00.000.00000 104 Transverse section through nasal cavity of adult gemfree rat000000000000.000.000.00.000 1'05 Purulent rhinitis in rat 20 days following aerosol inoculation with M;_pulmonis......... 105 Anterior ventrolateral nasal mucosa of adu1t gemfree rat0000000000000000000000 00000 106 Purulent rhinitis on floor of nasal cavity in rat 20 days following aerosol inoculation Wit-h€4—0Enlmonis0000000000.00.00.00.000000000 106 Colonies of M;_pulmonis growing on agar medium0000000000000000.000.000.000.0000000000 108 Colonies of M;_pulmonis growing on agar medium....................................... 108 xii Figure Page 43 Colony of M; pulmonis growing on agar medium.... 109 44 Colonies of M;_pulmonis on agar medium .......... 109 xiii INTRODUCTION Chronic respiratory disease (CRD) is the major problem affecting the health of the laboratory rat. It impairs the usefulness of the rat for most research since data obtained using rats afflicted with even the subclinical form of the disease are of questionable validity. In addition, CRD makes commercial production of dependable rats for research more costly and frustrating. It might also be considered inhumane to rear and maintain rats with this debilitating disease. As is evident from the literature, many terms have been applied to this disease. .For the purposes of clarity and convenience in this thesis it will be referred to as chronic respiratory disease. However, based on information obtained from this research, a new name will be proposed that is seemingly more specific and appropriate. Chronic respiratory disease is characterized by an insidious course which makes early detection of infection difficult. In fact, it may go undetected for many weeks by investigators using rats in their research. The incidence of infection in affected colonies, as determined by necrOpsy procedures, usually exceeds 50% and may reach 100%, and until recent years it was generally believed that most colonies were affected. During the past decade, however, some colonies apparently free of CRD have been established by utilizing a nucleus of parental stock derived by germfree techniques. The disease-free status is then maintained by rigid isolation and application of strict sanitary proce- dures. Although this approach has done much to reduce economic losses due to CRD and to improve the quality of the rat, the problem has not been entirely eliminated. While considerable attention has been devoted to the nature of this disease, the exact cause was unknown when this research was initiated. Only limited progress had resulted from research to determine the etiology and to experimentally reproduce the disease. A major problem in this regard was in obtaining rats that were clearly estab- lished to be free of the infection so that the condition could be reproduced at will. The germfree rat was a logical experimental animal to use in these investigations. The Department of Pathology at Michigan State Univer- sity had available a germfree animal facility, investiga- tors experienced in germfree techniques, and capable tech- nical assistants. Supportive facilities for pathologic and microbiologic research were also available. Chronic respiratory disease was present in several rat colonies maintained on the campus and these infected rats were attainable for investigation. Thus, the combination of expertise, facilities, and germfree and naturally diseased rats, was available for research to determine the cause and to experimentally reproduce CRD in rats of known microbial status. Previous to the time this investigation was initi- ated, germfree rats had not been used in experimental studies of CRD. My research on CRD began in December, 1968, and con- tinued the investigations of Dr. W. E. Giddens who worked and reported on the pathology and microbiology of the natural disease. He gave special attention to the gross and micro— scopic respiratory morphology of conventional, diseased and germfree rats (Giddens £2 31;, 1971a, 1971b). My research was directed toward identifying the causative agent and experimentally reproducing the disease in germfree rats with particular emphasis on the pulmonary aspect of the CRD syndrome. During 1969 and the early part of 1970, a series of transmission experiments was conducted. In a progress report (Burek, Jersey and Whitehair, 1970) of this research at a symposium on gnotobiotic research, mention was made that I had reporduced CRD with a mycoplasma isolated from the lungs of rats with the natural disease. Dr. R. G. Overcash, at the University of Alabama, who was attending the sympos— ium, contacted me in June 1970 and requested a supply of this organism. In order to verify the results of my experiments and to cooperate with other researchers on this disease, rats with the infection and broth cultures of Myc0p1asma pulmonis were supplied. A recent report from the group at the University of Alabama (Lindsey gt_al., 1971) verified the experimental reproduction of the disease. Following my appointment as instructor in clinical pathology (June, 1970) the research was continued but with reduced emphasis due to teaching and service responsibil- ities. LITERATURE REVIEW The literature on respiratory diseases of rats is rather voluminous but for various reasons much of it is not too pertinent to the problem today. In addition, it contains much repetition and duplication of earlier reports. Unfor- tunately, earlier workers often sought the cause of CRD strictly within the narrow perspectives of their particular areas of specialization. For example, bacteriologists sought to identify a species of bacteria, nutritionists a specific dietary component, and virologists a simple virus that caused CRD in rats. Although it would be unlikely that the cause of CRD when finally determined would be within the realm of any of these varied disciplines, these examples do illustrate the breadth of interest generated by an important disease of unknown cause. On the other hand, some of the literature resulting from the work of this variety of specialists did little but add to the confusion. It now appears that instead of a narrow disciplined approach to the problem, what was required was a coordinated multidisciplin- ary investigation. Primarily within the past decade, development and appli- cation of new research techniques, refinements in accuracy and precision of old techniques, advances in knowledge of infectious agents, and new concepts of pathogenesis have led to general acceptance of CRD as an infectious disease. Thus the tools were now available to re—evaluate certain widely accepted theories regarding its cause. In this literature review, while all available reports were carefully evaluated, for the sake of clarity and brevity, emphasis will be placed on critically reviewing only the more pertinent reports. Reports pertaining to the role of Mycoplasma pulmonis in CRD of rats will be stressed. In addition, some previous investigators have limited their research to either the rat or mouse but have made conclusions and formulated theories for both species. In this review, the two will be clearly separated because it may not be valid to assume the disease is the same in both rats and mice. Early Reports Klein (1903) reported the isolation of a previously undescribed diphtheroid bacterium from pneumonic lungs of white rats. The bronchi and alveoli of the pneumonic lungs were distended by fibrinopurulent exudate that contained masses of microorganisms. Subcutaneous abscesses were produced by injection of the organism into rats and guinea pigs. Although Klein's report was somewhat brief and lacking in details, it appears to be one of the earliest descriptions of CRD in the laboratory rat. Petrie and Macalister (1911) reported pulmonary disease in wild rats in England. They described the gross appearance of the lungs as follows: ”In the early stages, the lungs were found on incision to contain one or more small cavities, full of glairy, semipurulent material. In advanced stages, the normal lung tissue was replaced almost entirely by a system of large cavities full of thick caseous pus." Macalister and St. John Brooks (1914) reported similar observations in another group of wild rats captured in the same general area. Hektoen (1916) at the Memorial Institute of Infectious Diseases, Chicago, described "purulent or mucopurulent bronchitis and bronchopneumonia" in white laboratory rats and in one of 28 wild gray rats that were specifically examined for pulmonary lesions. He isolated a streptothrix from the diseased lungs which he believed was the same organ- ism described by Tunnicliff (1916). Two types of pulmonary consolidation were distinguished by Hektoen in the rats he examined. The first type was characterized by suppuration. Abscesses and dilated pus-filled bronchi were seen in the later stages of the bronchopneumonia. The second type was characterized by its gelatinous appearance due to "mucoid degeneration of the bronchOpneumonic exudate.” A photomicro- graph illustrating lesions of the gelatinous type depicted the junction of normal lungs and an area of the mucoid bronchOpneumonia. The illustration of mucoid bronchOpneu— monia represented what appeared to be a region of partial atelectasis in which mucus had collected in the partially collapsed alveoli. The clinical signs observed by Hektoen were: "lack of energy, standstill or loss of weight, ruffled hair, and failure to main- tain usual degree of cleanliness." These clinical signs are essentially the same as listed in current publications for rats with advanced lesions of CRD. Nelson and Gowen (1930), and Nelson (1930), at Rocke~ feller University, investigated the labyrinthitis occurring in rats of a large colony maintained at that institution. Their initial observations confirmed those of McCordock and Congdon (1924) but found in addition that many apparently normal rats suffered from otitis media although only a small percentage developed signs of inner ear dysfunction. The incidence of otitis media was 69% in adult rats (1 year of age or older) and 32% in young rats (3-4 months of age). Rhinitis and pneumonia commonly accompanied the otitis media in adult rats but only rarely in young rats. In order of decreasing incidence, Bacillus actinoides, streptococci and diphtheroids were most commonly isolated from the infected tympanic cavities. However no bacteria were recovered from the ears of 44% of the young and 10% of the adult rats although there was ample evidence of otitis media in all cases. The tympanic cavities of rats judged to be free of middle ear disease were negative for bacteria. Cultures of the nasopharynx and lungs gave similar results to those of the tympanic cavities. Intraaural inoculation of either g; actinoides or the streptococci in young rats resulted in a 75% incidence of otitis media whereas only 20% of the rats inoculated with the diphtheroid developed otitis media. 2; actinoides was recovered from 65% of those inoculated, streptococci from 10% and the diphtheroid was never recovered. Passey E; EL; (1936) described the gross and micro- scopic lesions of naturally occurring pneumonia in 251 laboratory rats. The rats were maintained on several different diets some of which were inadequate in vitamin A. Bronchiectasis was observed in 51% of all rats studied. The incidence of bronchiectasis did not seem to be significantly affected by diet. However, the incidence of squamous meta- plasia of the bronchial epithelium with keratinization was markedly increased in rats fed diets inadequate in vitamin A. Etiology of CRD The search to determine the primary cause of CRD in the rat has resulted in the implication of a variety of organisms and non-viable factors including bacteria, myc0plasmas, viruses, immunologic mechanisms, environmental conditions, and nutritional factors. Etiologic schemes involving ele— ments from more than one category have also been prOposed. This wide variety of proposed causes reflects the longstand- ing uncertainity that has surrounded the etiology of CRD. Current Status. Nelson (1946a) in a report entitled, ”Studies on Endemic Pneumonia of the Albino Rat," separated the chronic respiratory infection commonly observed in rats into two distinct but often intercurrent infectious diseases. 10 In later reports, Nelson (1948a, 1951, 1955, 1957, 1963, 1967) confirmed and repeated his earlier conclusions on the cause of CRD. As proposed by Nelson, infectious catarrh, caused by M; pulmonis, accounted for the upper respiratory and middle ear lesions, and endemic pneumonia (enzootic bronchiectasis, Nelson, 1957; chronic murine pneumonia, Innes gt 31., 1956), caused by a virus was responsible for the pulmonary aspect of the syndrome. This etiologic theory of CRD has gained general acceptance. The presence of the virus in pneumonic lungs of rats was determined by inoculating young mice with suspensions of pneumonic lung and exudates from rats and filtration experiments (Nelson, 1946a, 1946b). Joshi e£_al. (1965) reported isolation of a virus from pneumonic lungs of rats and reproduction of the pulmonary lesions of CRD in rats inoculated with the virus but, as pointed out by Giddens (1968), their conclusions were not well supported by their data. Notwithstanding the report of Joshi gt_al., the virus of CRD apparently has never been isolated. Kohn and Kirk (1969) and Lindsey et_al. (1971) reported experimental reproduction of the characteristic lesions of CRD in disease-free rats by intranasal inoculation of liquid cultures of M; pulmonis. Lutsky and Organick (1966) produced experimental respiratory infections in germfree mice using pure cultures of M; pulmonis. On the other hand, Bell and Elmes 11 (1969) failed to infect rats with cultures of ML_pulmonis and concluded that M;_pulmonis does not play a significant role in CRD in rats. Giddens (1968) concluded that prolonged and continuous irritation with accompanying antigenic stimulation of the bronchial wall was probably the primary cause of the pulmon- ary lesions of CRD. Brennan e£_al, (1969a) concluded that CRD was caused by the synergistic interaction of ML_pulmonis and Pasteurella pneumotropica. Clearly, the observations and conclusions of recent investigators has left the etiology of CRD in a state of uncertainty. The inability of investigators to isolate the virus proposed by Nelson (1946a) has stimulated considerable interest and further investigation. Mycoplasmas. The causative agent of contagious bovine pleuropneumonia, Mycoplasma mycoides, reported by Nocard and Roux (1898) was the first species in the genus, Myc0p1asma. Subsequently, numerous species have been isolated from a wide variety of sources including fowl, laboratory rodents, domestic and wild animals, man, sewage, soil and plants. The pathogenicity of several species has been firmly estab- lished whereas others appear to be commensals or saprophytes. Klieneberger (1935) reported an unusual organism in cultures of Streptobacillus moniliformis. She named them L organisms after the Lister Institute where she worked and, 12 noting their similarity to the agent of bovine pleuropneu- monia, described them as plueropneumonia-like organisms (PPLO). It was later discovered that the L organisms were growth variants of Streptobacillus moniliformis that lack a cell wall and have the potential to revert to the parent strain (Dienes, 1938). The term "pleuropneumonia-like," or simply PPLO, was adopted and in general use until supplanted by the current name of Mycoplasma (Edward and Freundt, 1956). Klieneberger - Nobel (1961) has written a well documented historical review of these organisms. Mycoplasmas are the smallest known free-living organ- isms. They have no rigid cell wall, are bounded by a limiting membrane and, except for one saprophytic strain, they require sterols for growth. Carter (1970) gave a con- venient summary of the major characteristics of the myco- plasmas and listed the more important pathogenic species of animals. Klieneberger and Steabben (1937) at the Lister Insti- tute, London, first reported isolation of an organism resembling the causative agent of contagious bovine pleuro- pneumonia from pneumonic lungs of rats. Designated the L1 organism and placed in the group of pleuropneumonia-like organisms (PPLO), it was recovered from 17 of 19 lungs with gross lesions of pneumonia. It was not cultured from any of 10 lungs that were free of macroscopic lesions. In a succeeding report (Klieneberger and Steabben, 1940), the L organism (changed from the previous desgnation 3 13 as Ll organism) was isolated from the lungs of one of 17 wild rats and 138 of 251 laboratory rats. Attempts to reproduce the disease with the L organism proved fruitless. 3 The authors' explanations were: "because so large a percentage of the stock rats harboured the organism and developed the characteristic lung condition that the ordinary routine of experimental infection was precluded." These were the same circumstances that have confronted other researchers working with these microorganisms in succeeding years. Nelson (1937a) reported an outbreak of infectious catarrh in a colony of Swiss mice. At necropsy, rhinitis, otitis media and slowly progressive pneumonia that usually terminated in death were characteristic of the disease. Chattering, a peculiar sound likened to the sound made by clicking the teeth together, was the only consistent clini- cal sign observed during the early course of the disease. Later in the disease loss of weight, rough hair coat, and labored respirations sometimes became evident. Intranasal inoculation of exudates obtained from the nose, middle ear or lung of diseased mice into normal mice was used to experimentally transmit the infection. The incubation period was usually about 10 days and the mortality rate high after 3 to 4 weeks. Nelson noted that the disease was highly communicable by direct contact, but that indirect contact was probably of little significance since in his experience normal mice held in cages in the same rooms with l4 cages of infected mice often remained uninfected for extended periods of time. In a second report, Nelson (1937b) described the detection and isolation of coccobacilliform bodies in the nasal, middle ear and pulmonary exudates from mice with infectious catarrh. The coccobacilliform bodies were observed in Gram-stained smears of the exudate as gram— negative ovoid or rod-shaped bodies. Ring forms with an unstained center were also seen on occasion. These bodies were seen within the amorphous debris and in cytoplasm of leukocytes found in the exudate. Pure cultures of the organism were obtained in tissue cultures. In a third report in this series on infectious catarrh of mice, Nelson (1937c) discussed the etiological signifi- cance of the coccobacilliform bodies. Pure cultures of the coccobacilliform bodies were infective for healthy mice upon intranasal instillation following as many as 12 passages of the organism in tissue cultures. Nelson concluded that the coccobacilliform bodies were very likely the cause of infect- ious catarrh in mice. Nelson (1940a) conducted 37 consecutive serial passages of infectious catarrh in rats over a period of 5 years by intranasal inoculation of nasal and middle ear exudates. Both Bordetella bronchiseptica and Actinobacillus muris (Streptobacillus moniliformis) were present in the first inoculum, but after the 12th passage neither organism could be recovered from the nasal passages or middle ear of the experimentally infected rats. However, the inflammatory 15 reaction remained unchanged in rats of all transmissions conducted after bacteria were no longer present in nasal and middle ear exudates. Nelson concluded that these bacteria had no causative role in infectious catarrh of rats. In a companion report, Nelson (1940b) described gram- negative coccobacilliform bodies that were demonstrable in nasal and middle ear exudates from rats with infectious catarrh. These coccobacilliform bodies resembled the agents of fowl coryza (Nelson, 1942) and mouse catarrh (Nelson, 1937b). The organisms could be cultivated in tissue cultures prepared from minced chick embryo suspended in Tyrode's solution. Intranasal inoculation of normal rats with cultures of the organism grown in tissue culture pro- duced the typical signs of lesions (rhinitis and otitis media) of infectious catarrh. The organisms could be demon- strated in stained smears of nasal and middle ear exudates obtained from the experimentally infected rats. The infec— tion was also transmitted by direct contact from the experi- mentally infected rats to normal rats that were housed in the same cage. Nelson noted that these coccobacilliform bodies resembled the pleuropneumonia group of organisms, but he was not sure they should be included in that group. He was unable to cultivate the coccobacilliform bodies in artificial media other than by the chick embryo tissue culture method. Coccobacilliform bodies (mycoplasmas) of similar appear- ance were observed in stained smears of catarrhal exudate 16 from the upper respiratory tract of chickens (Nelson, 1942), mice (Nelson, 1937b) and rats (Nelson, 1940b). Noting that in all 3 species the site of inflammation, type of inflamma- tory reaction, and appearance of the microorganism were strikingly similar, Nelson (1942) attempted to reciprocally transmit the infection among the 3 hosts. The diSease was not transmissible from either the rat or mouse to the chicken nor from the chicken to either of the rodents. However, reciprocal transmissions between the rat and mouse were readily accomplished. Nelson (1948b) noted a respiratory infection during the nasal passage of normal lung suspensions in mice. A pleuro- pneumonia-like organism (mycoplasma) was recovered from nasal, middle ear and pulmonary exudates. The disease was reproduced in mice by intranasal inoculation of suspensions of the exudates or lung and pure cultures of the mycoplasma. The infection was characterized by rhinitis, otitis media and pneumonia in the mice. Rats were also infected by inoculation of the exudates and in a single trial by inocu- lation of a pure culture of the mycoplasma. Rhinitis and otitis media but rarely pneumonia were found in the experi- mentally infected rats. The infection was transmitted in both mice and rats by direct contact. Nelson emphasized the importance of examining the middle ears and nasal passages of rats and mice when a myc0plasma infection was suspected. Pankevicus gt_al. (1957) isolated mycoplasmas from the pneumonic lungs of 14 of 25 rats. Rats free of respiratory 17 disease were inoculated intrabronchially with these myco- plasmas and passed three times in this manner. The myco- plasmas were recovered from the lungs of inoculated rats but lesions characteristic of chronic respiratory disease were not observed in the lungs. Pankevicus gt_gt. concluded that the role of mycoplasmas in the etiology of the pulmon— ary aspect of CRD was highly questionable. Joshi gt gt. (1965) isolated a mycoplasma from 30% of pneumonic rat lungs and 36% of "apparently normal" rat lungs. Mycoplasmas were isolated from nasal washings of 35% of rats with pneumonic lungs and from nasal washings of 32% of rats with apparently normal lungs. Lungs from 10 rats that had been inoculated intranasally with liquid cultures of myco- plasma were free of lesions on histologic examination. In a series of excellent reports (Lutsky and Organick, 1966; Organick gt gt., 1966; Organick and Lutsky, 1968; Lutsky and Organick, 1968), the pulmonary lesions caused by Mg_pulmonis in germfree mice were described. The methods employed to evaluate the experimentally infected mice included light, electron, and fluorescence microscopy, and cultural procedures. In addition, the thorough investiga— tions conducted by the authors to solve technical problems confronted during the research were informative for anyone utilizing germfree animals to investigate an infectious disease and especially for investigators interested in the role of Mt pulmonis in respiratory diseases of mice and rats. 18 In their first report, Lutsky and Organick (1966) reported the experimental production of gross lesions of pneumonia in 50 of 73 germfree mice that were killed or died between 2 hours and 4 weeks following intranasal inocu- tion with broth cultures of Mg_pulmonis. The microscopic lesions included peribronchial infiltration of lymphocytes and bronchi filled with polymorphonuclear leukocytes. Germ- free mice that were inoculated with Mt_pneumoniae or M; salivarium did not develop pulmonary lesions. In prelimi- nary eXperiments, 18% of conventional mice that were inocu- lated with sterile broth developed gross pneumonia. In contrast, of 66 germfree mice inoculated with sterile broth, none developed gross pulmonary lesions. By examination of lungs electronmicroscopically at one week following inoculation of Mt_pulmonis, the organisms were found to localize primarily at the surface of bronchial epi- thelial cells (Organick gt gt., 1966). Similar results were obtained from indirect fluorescent antibody tests (Organick and Lutsky, 1968). To determine the viability of the myco- plasmas in the isolator environment, 202 samplings of the isolator interior, food, water, litter, and feces were cultured for mycoplasmas, and all samples were negative (Lutsky and Organick, 1968). In some cases, the samples were obtained from cages that contained severely infected mice. Cheng (1954) reported that the bronchiectasis which followed bronchial ligation in young rats was attributable 19 to bronchitis and bronchial stagnation. However, the cause of the bronchitis in the ligated lung remained uncertain, since bacterial pathogens were not isolated from the bronch— ial exudate. Klieneberger-Nobel and Cheng (1955) isolated the pleuropneumonia-like L3 organism in heavy growth from the bronchial exudate of rats killed 3 to 12 days following ligation of a bronchus. The organism was recovered from the lungs of one of three rats in which a bronchus had not been ligated. It was postulated that accumulation of bronchial exudate following ligation permitted multiplication of the L3 organism leading to infection of the bronchus. In turn, the weakened bronchus would dilate under pressure of the accumulated exudate. The L3 organism was later identified as M; pulmonis (Klieneberger-Nobel, 1961). Ventura and Domaradzki (1967a, 1967b), physicians at St. Joseph of Rosemont Hospital, Montreal, studied the effects of bronchial ligation in rats. An occluding perman- ent stricture of the bronchial lumen usually resulted in accumulation of mucopus and bronchitis leading to bronchiec— tasis. They found that bronchiectasis developed only when the lungs was infected by Mygoplasma pulmonis. In atelec- tatic lungs negative for mycoplasma there was hypersecretion of mucus but no bronchial inflammation. Bronchial ligation with subsequent removal of the stricture led to resolution of the inflammatory changes and disappearance of bronchiec- tasis in some cases. With longer periods of total occlusion prior to re-establishment of bronchial drainage a smaller 20 percentage of cases was resolved. However, as the interval between removal of the ligature and subsequent examination was increased the percentage of recoveries also increased. It was concluded that both bronchial occlusion and an infec- tion were required to produce bronchiectasis. In addition, the authors concluded that the mycoplasma infection was directly related to the inflammation of the bronchus. Brennan gt g1. (1969a) at Argonne National Laboratory, studied pneumonia caused by M; pulmonis and Pasteurella pneumotropica in conventional, specific pathogen-free (SPF), and germfree mice. They were able to produce pneumonia in conventional and SPF mice only when both organisms were present. Pneumonia was produced in germfree mice by inocu- lation of the organisms singly, but they concluded that the pulmonary lesions more closely resembled those seen in the naturally occurring disease when the organisms were present simultaneously. In a succeeding report, Brennan gt_gt. (1969b) reviewed the etiologic agents variously associated with "murine pneu- monia." They defined "murine pneumonia" as "any pneumonia described in mice and rats" and concluded that Mt_pulmonis and E; pneumotropica were the usual causes. They further concluded that concurrent infections with these organisms resulted in fulminating pneumonia and death. Kohn and Kirk (1969) at the West Virginia University Medical Center reported the experimental production of typical pulmonary lesions of CRD in rats with pure cultures 21 of Mycgplasma pulmonis. They used conventially reared progeny of gnotobiotic rats in experiments of seven months duration. This report did not attract too much attention at that time because of the currently accepted viral etio- logy of the pulmonary lesions of CRD in rats. In retro- sepct, Kohn and Kirk should receive due credit for their original work. In two series of experiments, Kohn and Kirk, exposed a total of 68 rats to 4-day broth cultures of Mt pulmonis either directly by serial inoculation at 7-21 days of age or indirectly by housing in adjacent or in the same cages. They had originally isolated the culture from the lungs of diseased rats and identified the organism by the growth inhibition test. Nine adult breeders were included in the experiments. The rats were maintained in "pathogen con- trolled" quarters until the young reached seven months of age at which time they were killed and examined. Cultures of the middle ear, nasal turbinates, throat and lungs resulted in recovery of Mt_pulmonis from 58 of the 68 rats. Pulmonary consolidation and bronchiectasis were observed grossly in 14, and two had "greatly increased peribronchial lymphoid hyperplasia" on microscopic examina- tion. Mt pulmonis was recovered from the lungs of all rats with discernible pulmonary lesions and additionally from 7 without pulmonary lesions. Cultures for bacteria from the same sites as cultured for Mt_pulmonis resulted in isolation of Streptoccus viridans, nonhemolytic streptococci, 22 micrococci, Staphylococcus epidermidis, Escherichia coli, diphtheroids, and Proteus mirabilis. Not all rats were cultured for bacteria. §; viridans and micrococci were the only bacteria isolated from the lungs. No bacteria were recovered from the middle ear. Bell and Elmes (1969) at Queen's University, Belfast, concluded that §;_moniliformis and Mt pulmonis "do not play an important role in the etiology of CRD." They found that most conventional rats (3 to 18 months of age) harbored §; moniliformis and Mt pulmonis in the nasopharynx and some- times in the lungs, and that most rats had histologic lesions of CRD. In contrast, SPF rats of similar age were free of both the microorganisms and microscopic lesions of CRD. Experimental inoculations with 6-day broth cultures of Mt pulmonis, 3-day cultures of §; moniliformis, a mixture of the 2, or sterile broth did not cause CRD in SPF rats inocu- lated at 4 weeks and killed at 10 months of age. However, typical lesions of CRD were produced by inoculation of a suspension of pneumonic lung from which both Mt_pulmonis and S; moniliformis had been isolated. In a subsequent experiment, rats were inoculated with a suspension of pneu- monic lung from which Mt_pulmonis and §t_moni1iformis could not be isolated, and these rats also developed the lesions characteristic of CRD. Bell and Wheeler (1970) reported on the susceptibility of caesarian-derived (C-D) rats to natural infection with 23 CRD. They considered the cause of CRD to be a mycoplasma and gray-lung virus. Young C-D rats were found less suscept— ible than adult C—D rats to CRD. Young C-D rats more readily contracted the infection when eXposed to adult con- ventional rats than when similarly exposed to young con- ventional rats. Caging of C-D rats just 4 feet from infected conventional rats significantly reduced the inci- dence of CRD in the C-D rats when compared to the incidence of CRD in C-D rats that were housed in the same cage with conventional rats. The authors concluded that young C—D rats can be mixed with young conventional rats for 2 months with little risk of developing CRD. Kohn (1971a) described the sequential pathogenicity of Mt pulmonis in 136 weanling rats that were examined from 1 to 24 weeks following inoculation of 4-day broth cultures of Mt pulmonis. The organism was reisolated from 92% of the inoculated rats but from none of a like number of con- trol rats. Lesions observed in the inoculated rats were otitis media in 98 rats, tracheitis in 17 rats, and bronch— iectasis in 4 rats. Evaluation of peribronchial lymphoid accumulations revealed a nearly 3-fold increase in the infected when compared to the control rats. In a companion report Kohn (1971b) described the elec- tron microscopic lesions in bronchiectatic lungs of rats infected with Mt_pulmonis. The mycoplasma were usually located at the cell membrane surface and occasionally within cytoplasmic vaculoes of bronchial epithelial cells. Other 24 ultrastructural lesions were loss of cilia and vacuoliza- tion of the cytoplasm in epithelial cells. Lindsey gt gt. (1971) inoculated disease—free rats with broth cultures of Mt_pulmonis and concluded from their results that Mt pulmonis acting independently was capable of causing the characteristic syndrome of CRD in rats. The experimental disease was characterized by otitis media, rhinitis, tracheitis and regional pulmonary consolidation. Microscopic pulmonary lesions described as epithelial hyper— plasia, peribronchial lymphocytic infiltration and bronchial luminal accumulation of purulent exudate were considered primary tissue alterations. Secondary changes included con- solidation of alveoli with neutrophils and macrophages, bronchiectasis, atelectasis, squamoid changes of the bronch- ail epithelium, formation of peribronchial acinar Spaces and pulmonary abscesses. Especially pertinent to the research presented in this thesis was markedly greater pathogenicity of their Michigan (M) isolate compared to 4 other isolates that were tested. The M isolate was supplied to Lindsey gt g1. by the author. Lindsey gt_gl, considered pulmonary abscesses as the most severe pulmonary lesion observed in the experimentally infected rats. Pulmonary abscesses developed only in rats that had been inoculated with M isolate. Lindsey gtht, suggested the name "rodent pulmonary myc0plasmosis” would be more appropriate for the syndrome of CRD if future investigations confirmed their conclusions. This name, however, would seem to over- 25 emphasize the pulmonary aspect of the syndrome, since lesions were observed throughout the respiratory tract. Viruses. Nelson (1946a) reported the transmission of "endemic pneumonia”, which he considered distinct from infectious catarrh, from infected rats to mice by intra- nasal inoculation of suspensions prepared from the lungs of rats with the disease. Although otitis media was seldom observed in the rats used as a source of infectious material, exudate from one with middle ear infection was used to inoculate mice and caused the same disease as when lung was used as a source of inoculum. The disease produced in mice was characterized by bronchOpneumonia resembling the early stages of endemic pneumonia in the rat but differed in the later stages in that bronchiectasis did not develop as it does in rats. The disease in mice also differed in that, unlike rats, mice usually developed otitis media. Like the rat, the mice developed only a low grade rhinitis with scanty exudate. The disease was transmissible by direct contact but only after prolonged cohabitation of the unin- fected mice with infected mice. Nelson noted that the disease closely resembled infectious catarrh, but neverthe- less he considered it a distinct disease. In a companion report Nelson (1946b) described the nature of the causal agent of endemic pneumonia of rats as determined experimentally in mice. No single species of bacteria was consistently isolated from the experimentally 26 infected mice. Concerted attempts to recover a pleuropneu- monia—like organism by cultural methods were unsuccessful and the organism was not seen in stained films of exudates. Lung suspension in dilutions to 105 were regularly infective for mice and occasionally dilutions of 107 were infective. The higher dilutions often produced only otitis media in the inoculated mice. The organism passed Berkefeld V but not N filters. Centrifugation for 30 minutes at 9000 r.p.m. resulted in a noninfectious supernatant in 2 out of 3 trials. Attemtps to cultivate the agent in embryonating hen's eggs were unsuccessful. Infective lung suspensions were inactivated by refrigeration at 40F for 1 week. The agent remained viable when stored under dry ice for up to 13 weeks. Nelson concluded that his studies did not unequivocally identify the agent of endemic pneumonia as a virus but that it should rather be regarded as virus-like. Nelson (1948a) attempted experimentally to infect young rats (1 to 2 months old) with the virus-like agent of endemic pneumonia (Nelson, 1946b) but found that young rats were refractory to the infection. Experimental infections of adult rats were not practical since a very high percent- age of rats in the colony were naturally infected. In pursuing this problem Nelson found the rats in his colony usually acquired the infection during the suckling period and carried the infection in their nasal cavities or other parts of the respiratory system throughout life. He reached these conclusions by inoculating young mice with 27 washings from the nasal cavity or with lung suspensions from rats of various ages. Nelson (1949a) reported the recovery from the lungs of 2 wild rats of a pneumotropic virus which was readily trans- mitted to laboratory mice and rats by intranasal inoculation of lung suspensions. The disease produced in mice was characterized by rapid onset, high mortality and an infiltra- tive penumonia with pulmonary edema. Mice also occasionally developed otitis media. Rats infected intranasally with mouse lung suspensions were similarly affected, but the pneumonia was less acute than that produced in mice. Trans- mission by direct contact was possible in both species but somewhat irregular and required an exposure period of at least 3 weeks. Nelson considered this disease distinct from edemic pneumonia of rats and mice and named it wild rat pneumonia (WRP). In a companion report, Nelson (1949b) described the biological characteristics of the WRP agent. A causative bacterium or pleurOpneumonia-like organism could not be demonstrated by cultural and microscopic examinations. Lung suspensions were not inactivated by addition of penicillin and streptomycin. Transmission experiments resulted in 2 distinct types of pulmonary reaction in mice which suggested the presence of more than one causative agent, since the respective types could be serially transmitted in mice. Nelson noted that the typical lesions of endemic pneumonia 28 occurred only in mice killed 4 weeks or more after inocu- 1ation. He apparently did not consider the possibility of these lesions being simply the later stages of the same pathological process. The results of several sedimentation and filtration experiments were rather confusing, but Nelson was of the Opinion they tended to support his hypothesis of a dual in- fection. He concluded WRP most closely resembled grey-lung disease of mice (Andrews and Glover, 1945). Grey-lung virus (Andrews and Glover, 1945) was isolated from mice and named according to the gross appearance of the pulmonary lesions. Both the agent and lesions persisted indefinitely in infected mice, and deaths occurred only rarely. The early inflammatory changes were characterized by exudation of neutrophils. At 3 weeks and persisting for up to 10 months, perivascular and peribronchial lymphocytic cuffing were prominent microscopic lesions. Andrews and Niven (1950) described lesions in cotton rats which they attributed to the grey-lung virus. They reported the disease was cured by treating infected animals with chlor- tetracycline or oxytetracycline, but not by treatment with sulfonamides and penicillin. Andrews (1964) concluded that, on the basis of antibiotic sensitivity, the grey-lung virus was probably a mycoplasma. Vrolijk gt gt. (1957) reported the demonstration of grey-lung virus in laboratory and wild rats by intranasal inoculation of mice with suspensions of rat lungs. Electron- 29 microscopic examination of partially purified lung suspen- sions revealed "virus-like particles having mean diameter of approximately 200-250 mu." Marmion and Hers (1963) noted some similarities between M; pneumoniae and grey-lung virus. They suggested that grey- 1ung virus might instead be a mycoplasma. Gay and Attridge (1967) described the fine structure of an agent in the lungs of mice that was susceptible to tetracyclines. The mice had been infected by intranasal inoculation of suspensions of pneumonic lungs from rats. Gay and Attridge noted that both this agent and grey-lung virus were sensitive to tetra- cyclines. Gay (1967) compared the fine structures of the gray-lung virus and the "rat pneumonia agent" and concluded that they were essentially identical. Both agents were sensitive to tetracyclines but resistant to sulfonamides, streptomycin, penicillin and chloramphenicol. Gay called them "mycoplasma-like" agents but was unable to isolate them by standard mycoplasma cultural techniques. Bacteria. Numerous reports appear in the early literature concerning the bacterial flora of the respiratory system of rats. Most of these early investigations grew out of the concern for diseases transmissible from rats to man. Later, reports appeared because of interest in the etiology of respiratory disease in rats. Based on the opinions of Giddens (1968) and Nelson (1967) and my review of the literature, it appears that bacteria have little or no 30 significance as a primary cause of CRD in the rat. There— fore only a brief review of the literature on bacteria is presented here. In their review of the literature, Klieneberger and Steabben (1937) noted that no single species of bacterium had been regularly isolated from the lungs of rats with pneumonia even though the pulmonary lesions described by the various authors were generally quite similar. In fact, no organism at all was reported in many cases. Species of bacteria commonly isolated from the res~ piratory tract of rats include Streptobacillus moniliformis (Tunnicliff, 1916; Jones, 1922; Nelson, 1930; Strangeways, 1933; Klieneberger and Steabben, 1937; Bell and Elmes, 1939), Bordetella bronchiseptica (Hoskins and Stout, 1919; Jones, 1922; Smith gt gl., 1930; Pankevicus gt gt., 1957; Winsser, 1960; Burek, 1970), Pasteurella pneumotropica (Jawetz, 1950; Jawetz and Baker, 1950; Hoag gt gt., 1962; Heyl, 1963; Flynn gt gt., 1965; Wheater, 1967; Giddens, 1968; Brennan gt_gt., 1969b; Burek, 1970), Diplococcus pneumoniae (Mirick gt gt., 1950; Innes gt gl,, 1956; Ford, 1965; Baer, 1967; Giddens, 1968; Weisbroth and Freimer, 1969), Corynebacterium kutscheri, (Kutscher, 1894; LeMaistre and Thompsett, 1952; Giddens, gt gt., 1968). Giddens (1968) gave a comprehensive review of the literature on the cateria that have been isolated from the respiratory tract of rats. He surveyed the respiratory tracts of CRD-affected rats from three different sources for 31 bacteria and found that a great variety had been isolated. The bacteria most frequently isolated were Dt_Pneumoniae, P; pneumotropica, Staphylococcus aureus, Micrococcus sp., and C;_kutscheri. Giddens found no correlation between the bacteria isolated and lesions found. Brennan gt_gt. (1969a) studied chronic pneumonia in mice and concluded that it was caused by infection with Mt pulmonis and E; pneumotropica acting synergistically. They extrapolated their data and applied the same conclusions to the rat. Burek (1970) inoculated young conventional and germfree rats with aerosols of Pt_pneumotropica. No gross or microscopic lesions developed in the inoculated rats. Bordetella bronchiseptica was the organism isolated most commonly from pneumonic and grossly normal lungs of rats by Pankevicius gt g1. (1957). Burek (1970), produced pneumonia in conventional and germfree rats by inoculation with aerosols of B; Bronchiseptica. At one week following inoculation the rats were anoretic, dyspneic, and had rough hair coats. On gross examination of the lungs, there were tiny grayish foci measuring 1 to 3 mm in diameter dissemi— nated throughout the lungs. Microscopically, these focal lesions consisted of bronchopneumonia. The foci of broncho- pneumonia were regressing or had disappeared in rats killed 4 to 8 weeks after exposure. In rats inoculated simultane- ously with §t_bronchiseptica and P; pneumotropica, no differ- ence was observed from rats inoculated with §t_bronchiseptica alone. 32 Natural outbreaks of pneumonia in rats caused by E; pneumoniae have been reported by Mirick gt gt. (1950), Innes gt gt. (1956), Ford (1965), Baer (1967), and Weisbroth and Freimer (1969). Following several outbreaks of pneumo- coccal pneumonia in rats, Weisbroth and Freimer (1969) obtained 10 rats from each of 22 commercial sources located in northeastern United States. Diplococcus pneumoniae was isolated from the nasal passages of 156/190 rats from 19/22 of the commercial sources. Specific pathogen free rats did not harbor the organism. Streptobacillus moniliformis was at one time a common isolate from the nasal passages of laboratory rats in the United States but the incidence of this organism has decreased in recent years (Brennan gt_gt,, 1969b). Although E; moniliformis has not been established as a pathogen for rats, it has been well established as the cause of one form of rat-bite fever in man, and individuals working with rats should be familiar with this organism (Nelson, 1967). Giddens gt gt. (1968) reported a spontaneous outbreak of pneumonia caused by gt_kutscheri in a commecial rat colony. CaseOpurulent foci were observed in the lungs and occasionally in kidneys, liver and subcutis. Outbreaks of 'Ct_kutscheri infection, also known as pseudotuberculosis (LeMaistre and Thompsett, 1952), usually have occurred in rats under conditions of stress such as treatment with cortisone (LeMaistre and Thompsett, 1952) or vitamin deficien- cies (Zucker gt_gt,, 1956). Rats may harbor gt_kutscheri 33 without ill effects and then manifest the infection when stressed during an experiment. Clinical Signs Hektoen (1916) noted that rats with mild pulmonary disease could not be recognized clinically and concluded that pulmonary disease probably was more prevalent than was generally recognized at that time. Similarly, Innes gt gt, (1956) concluded that "macrosc0pic appearances alone may not lead to the detection of this disease" in laboratory rats. Klieneberger and Steabben (1937) found that rats without gross pulmonary lesions were rare but lungs without micro— sc0pic lesions were even less common. Clinical signs observed by Hektoen (1916) in rats with severe pulmonary lesions were lethargy, failure to gain weight or loss of weight, rough coat and failure to maintain the usual degree of cleanliness. Klieneberger and Steabben (1937) observed snuffling in rats and suspected it was an early sign of the disease. Later signs were described as deep labored breathing that included noticeable movement of the flanks. Innes gt gt. (1956) described unthriftiness, rough hair coat, loss of weight, snuffles, and wheezing in advanced stages of pulmonary involvement. Lindsey gt gt. (1971) gave the most comprehensive accounting of the clinicopathologic syndrome including the ages at which the various aspects of the syndrome were likely to occur in rats of a diseased colony. They rarely observed 34 CRD in rats less than 3 weeks of age but at l to 2 months of age snuffling and scanty encrustations about the external nares were observed. Clinically inapparent otitis media was observed in a large percentage of the rats and a few had manifestations of inner ear disturbance. Variable numbers of rats at 2 months of age had gross pulmonary lesions. The pulmonary lesions were often clinically silent at this time. Occasionally sudden epizootics of severe pulmonary disease were observed. Characteristic signs of severe pulmonary involvement included polypnea, inactivity, humped posture, rough coat and reduced weight gains. Mortality usually remained low. Pathology The morphologic pathology of naturally acquired CRD in the rat has been reported by Nelson and Gowen (1931), Hektoen (1915-1916), Passey gt gt. (1936), Klieneberger and Steabben (1937), Innes gt gt. (1956), Newberne gt gt. (1961), Innes gt gt, (1967), Nelson (1967), Giddens (1968), and Giddens gt gt. (1971a, 1971b). This is not an exhaustive list but is a representative sampling of the many reports in the literature. Since the pathologic descriptions in these reports often vary only in minor details or use of termino- logy, only selected reports are cited. Hektoen (1916) distinguished 2 types of gross lesions in the lungs. In the most common type, parts of l or more lobes were solid and red with reddish-yellow or yellow foci 35 that on the cut surface represented accumulations of cellular exudate within bronchi. Variations on this first type involved consolidation of entire lobes sometimes with much mucus in the bronchi or, in later stages, with multiple abscesses. In the second type, the lesions had sharp but irregular margins and appeared homogeneous, semi-translucent, light-grey and reddish—gray in color often with yellow foci. Lesions of both types were described in a single lobe or in some cases mixed lesions representing both types were observed. Klieneberger and Steabben (1937) noted the marked varia- tions in the gross appearance of the lesions and emphasized the fact that microscopic lesions may be well advanced with- out detectable macroscopic lesions. Passey gt_gt, (1936) noted mild localized dilatations of the bronchi that could be palpated in fresh specimens as small "shotty" nodules. When a nodule was incised, clear mucinous material oozed from the cut surface. Later lesions were gray or yellow nodules at the surface that represented "enormously dilated bronchi filled with caseous-like material." The cut surface of the nodules was dry and resembled tuberculous nodules. Innes gt gt. (1956) described entire lobes that were indurated, shrunken, rubbery in consistency and cobbled at the surface. The low incidence of empyema and pleural adhesions was emphasized. The lesions were uniformly distributed among the pulmonary lobes. Newberne gt_gt. (1961) noted lesions most commonly in the cardiac lobe followed by right apical 36 and apical portion of left lung. Other parts of the lungs were equally affected. Nelson and Gowen (1931) and Nelson (1967) described yellowish purulent exudate in the tympanic cavities of diseased rats. Nelson (1967) was the only author to mention compensatory hypertrophy of the unconsoli- dated portions of the lungs although several authors des- cribed compensatory emphysema in their microscopic descrip— tions. Klieneberger and Steabben (1937) viewed the earliest microscopic lesions in the lung as a complex set of con- current changes involving the bronchi. Hypersecretion of mucus, proliferation (hyperplasia) of the bronchial epithe— lium, and peribronchial lymphocytic infiltration led to accumulation of mucus and ultimately to dilation of the bronchi. As the bronchi expanded, the surrounding alveoli were collapsed. Neutrophils had begun to infiltrate through. the bronchial epithelium into the lumen while mononuclear cells were accumulating in the lamina propria and submucosa. With the passage of time the neutrophil—laden bronchial exudate increased to occlude the lumen. Finally there was necrosis of the neutrOphils and of bronchial epithelium and all that remained was a mass of pus surrounded by fibrous tissue. Newberne gt gt. (1961) observed the initial peribronch- ial accumulations of lymphocytes along the proximal portions of the bronchi. Innes gt gt. (1967) concurred with the observation of Newberne gt gt. (1961) but added that 37 perivascular accumulations of lymphocytes occurred simul- taneous to the bronchial lesions. Large numbers of eosino- phils were often associated with the perivascular accumula- tions in young rats. The peribronchial accumulations of lymphocytes often reached extensive proportions and primary follicles were common (Innes 22.21:! 1956; Newberne gt_gt., 1961). Innes gt gt. (1956) concentrated their description on changes in the walls of the bronchi and bronchioles giving only brief descriptions of the purulent component and the changes that take place in the bronchial epithelium. They described segmental bronchiectasis during the early stages reminiscent of the ”shotty nodules" palpated by Passey gt gt, (1936). Bronchiectasis generally progressed distally along the bronchial tree thus bronchiolectasis appeared during the advanced stages of pulmonary involvement. Peribronchial fibrosis followed the inflammatory and degenerative changes in and around the bronchial walls (Newberne gt gt., 1961; Innes gt gt,, 1967). Interstitial accumulation of inflammatory exudates in alveolar walls located adjacent to bronchi commonly accom- panied the bronchial changes in segments that were actively involved in the inflammatory and degenerative processes leading eventually to bronchiectasis (Innes gt_gt., 1956; Newberne gt gt., 1961). Occasionally the alveolar walls of entire pulmonary lobes were thickened in this manner (Innes gt gt., 1956). Peribronchial alveolar collapse was described 38 by all of the authors. Innes gt gt. (1956) commonly observed foamy macrophages within the alveoli, some containing pigment, but were uncertain of their significance since collections of these cells were often seen in otherwise normal lungs. They seldom observed filling of alveoli, stating that broncho- pneumonia was infrequent. Passey gt gt. (1936) expressed interest at observing bronchi markedly distended with exu- date yet the adjacent alveoli were uninvolved. They noted that as the purulent character of the bronchial contents increased the tendency for alveolar consolidation increased. This observation was especially apparent at the termination of bronchioles and gave rise to localized patchy broncho— pneumonia. Passey gt gt. (1936) observed squamous metaplasia of the bronchial epithelium but found marked accumulations of keratinized debris occurred only in rats on vitamin A deficient diets. Squamous metaplasia with minimal kera— tinization was rarely observed in rats on vitamin A adquate diets. Newberne gt gt. (1961) reported squamous metaplasia was a prominent finding and that keratinization was not uncommon. In contrast, Innes gt gt. (1956) reported Squamous metaplasia was uncommon in their experience. Newberne gt gt. (1961) described intervening areas of cxolumnar epithelium bordered by squamous epithelium and £$Uggested these may have represented a reversion to normal (RDlumnar epithelium. They noted proliferation of the bronch- jfiil epithelium was common and in some cases reached the 39 stage of papillary adenomatous hyperplasia. Compensatory emphysema in uninvolved areas of the lung was described by Passey gt gt. (1936) and Newberne gt_gt, (1961). Nelson and Gowen (1930) reported diagnosis of otitis media by the presence of thin mucoid or thick purulent exudate in one or both tympanic cavities. Neutrophils and occasionally smaller numbers of mononuclear cells were pre— sent in stained smears of the exudate. In some cases the lining of the tympanic cavity was thickened. Occasionally the tympanic membrane bulged outwardly although ruptured membranes were not observed. The osseous tympanic wall was softened and readily punctured with a pointed instrument. Involvement of bone was especially prevalent in rats with dysequilibrium. McCordock and Congdon (1924) described the histologic appearance of otitis media in rats. The tympanic cavity was filled with purulent exudate and proliferated fibrous con— nective tissue replaced the normal simple squamous epithelium lining of the middle ear. The inflammatory reaction often extended to the bone surrounding the cavity causing lysis and regeneration of the osseous tissue. Exudate was observed in the eustachian tube and nasal cavity. They postulated taxtension of the infection from the nasal passage along the (Eustachian tube into the middle ear cavity. Giddens (1968, 1971a) described small collections of rNautrOphils, edema of the lining, and metaplasia of the (Lining epithelium to ciliated columnar epithelium with 40 plentiful goblet cells in mild cases of otitis media. More severe cases were characterized by epithelial necrosis, purulent exudate filling the tympanic cavity and later granulation tissue replaced the pus. Cystic glands lined by simple cuboidal epithelium and filled with mucous or serous exudate were observed within the mass of granulation tissue. The inner side of the tympanic membrane was thickened. The auditory ossicles were often surrounded by connective tissue or by a pool of neutrophils. Giddens (1971a) described purulent rhinitis in the rat characterized by purulent exudate within the nasal cavities, diffuse subepithelial infiltration of lymphocytes and alter- ations of the epithelial lining that included necrosis in some cases . Respiratory Disease Control Nelson and Gowen (1931) established a rat colony which was free of middle ear disease by selecting only the progeny from parental stock that was free of the disease. Once an initial breeding stock was established, close inbreeding was Practiced for 7 generations at which time random mating was instituted. From the outset and throughout the period of idibreeding, the parents of each generation were killed and encamined for evidence of middle ear infections. 'The entire Smelected colony was reared in separate isolated quarters ‘mflay from the stock colony of origin. However, the incidence Of: pneumonia remained very high (52%) in adult rats from the 41 selected colony. Nelson (1940a) examined 200 rats from the selected colony and reported an 8% incidence of otitis media. Nelson (1951) reported the use of cesarean-derived and hand-fed parental stock to establish a colony of CRD-free rats. Although both infectious catarrh and "endemic" pneu- monia were present in the colony from which the parental stock originated, CRD was eliminated in the derived colony. The method used to establish the CRD-free colony was to maintain the progeny and subsequent generations in rigidly isolated quarters without introduction of outside rats into the colony. Foster (1958) established a CRD-free colony starting with cesarean—derived offspring foster-suckled by germfree mothers. The CRD-free colony was maintained behind a system of barriers to prevent reinfection by airborne or other routes. This general procedure has since been used on numerous occasions to establish CRD-free colonies. Ganaway and Allen (1969) housed rats in cages fitted with a filtering system by which the air supplied to each cage was filtered by application of "slight negative pressure." Sodium sulfamerazine was added to the drinking water at a rate of 1 gram per gallon of water. Examination of 56 off— spring from the original breeders revealed no evidence of respiratory disease. The offspring ranged in age from 3 weeks to 14 months of age at the time of examination. Ganaway and Allen concluded that sulfamerazine prophylaxis was respon- sible for elimination of respiratory disease. Haberman EE.§£° 42 (1963) reported the additon of sulfamerazine to the diet at the rate of 5 milligrams per 20 grams of diet eliminated respiratory disease from rats in the third through tenth generations. Chlortetracycline added to the diet at the same levels failed to eliminate respiratory disease. Summary of Literature Review It is evident from this literature review that CRD has attracted much attention in recent years. While the patho- logy of the natural disease has been well described, there still exists uncertainty as to the primary cause and etio- logic interrelationships of the disease. This uncertainty regarding the cause of CRD is quite apparent from the con- tradictory conclusions reached by Giddens (1968), Bell and Elmes (1969), Brennan gt gt. (1969), and Kohn and Kirk (1969) from their independent investigations. Clearly, there is need for additional research to clarify the cause of CRD and to further evaluate the well established etiologic scheme proposed by Nelson (1946a). OBJECTIVES The general objective of this research was to obtain additional information on chronic respiratory disease of the rat. In pursuing this general objective many avenues of investigation could have been followed. In the interest of making maximum use of available resources, my research inter- ests, and the aspect of CRD most deficient in knowledge, the specific aims were: 1. To experimentally reproduce CRD in the rat with a known pathogenic agent isolated from rats with the natural disease. To reisolate and identify the agent producing the disease. To describe the clinical signs and gross and micro- scopic pathology of the disease produced under Objective 1. To compare the nature of the lesions produced under Objective 1 with those of the natural disease. To evaluate critically the information from the above objectives and make recommendations for prevention and control of CRD in the laboratory rat. 43 MATERIALS AND METHODS This research was initiated in December 1968 and continued through June, 1972. Room 345 Giltner Hall, Barn 5 at the Veterinary Research Farm, and the Clinical Micro- biology Laboratory at the Veterinary Clinic were the primary facilities used to conduct these experiments. Experimental Rats Conventional rats affected with naturally acquired CRD (hereafter referred to as CRD-rats), germfree rats and ex— germfree rats were used in this research. "Ex-germfree" is the term currently applied to rats (animals) that are initi- ally reared under germfree conditions and then changed to a conventional environment (Gordon and Pesti, 1971). CRD-Rats. Approximately 150 CRD-rats were obtained to study the clinical and pathological manifestations of the natural disease, to use in natural transmission experiments, and to provide a source of inoculum for aerosol transmission experi- ments. Sources of CRD-rats were three conventional colonies maintained on the Michigan State University campus and two commercial colonies located in the State of Michigan. In each of these colonies, evidence of CRD had been prevalent for a number of years. 44 45 CRD-rats obtained from the on-campus colonies were being discarded due to illness (respiratory disease), advanced age, or following termination of an experiment. Rats from the commercial colonies were obtained during an earlier investi- gation (Giddens, 1968) and had been housed for several months in room 345 Giltner Hall. As they became available, rats from on-campus colonies were intermingled with rats from the commercial colonies in room 345. This room was not an ideal environment for housing rats becuase of variations in tem- perature, poor ventilation, dampness, and uncontrolled lighting. Furthermore, these deficiencies of design and equipment were complicated by over-crowding, use of the room to house several different Species, and poor sanitation. It was, however, a suitable location to house the CRD-rats since progressive development of their respiratory syndrome was desirable. Germfree Rats. Germfree rats were used for exposure to a cell-free filtrate of lungs from the CRD-rats (Experiment 4) and to isolated cultures of Mt pulmonis (Experiments 5 and 7 to 11). These rats were from a colony maintained at Barn 5 that had been established from rats originally of the Sprague-Dawley strain.* In addition, germfree rats of Notre Dame-Wistar stock were available during the prelimin- ary stages of the research but later became contaminated with bacteria. *The A. R. Schmidt Co., Inc., Madison, Wisconsin. 46 Egjgermfree Rats. Ex-germfree rats used for natural trans— mission eXperiments were the contaminated Notre Dame-Wistar rats (Experiments 1 and 3) and rats of the Sprague—Dawley strain (Experiment 2) from an isolator that had become contamin- ated with bacteria. Ex-germfree rats used for the aerosol transmission experiment that was ocnducted under conventional housing conditions (Experiment 6) were obtained by removing germfree rats from the isolators maintained at Barn 5. Experiments A series of eXperiments was conducted to experimentally reproduce CRD in rats and identify the causative agent. The initial experiments were on natural production of CRD by exposure of clean (ex-germfree) rats to diseased rats. These experiments were then followed by experiments to pro- duce the disease by aerosol inoculation of germfree and ex- germfree rats of various ages. The routine procedures used throughout these experiments such as rations, germfree tech- niques for isolation and growth of Mt_pulmonis, and gross and microscopic examination of tissues will be given later. For convenience in presentation of data some details of procedures will be included in the results. Exposure to Natural Disease. In a series of 3 experiments, a total of 50 ex-germfree rats was placed in Room 345 Giltner Hall where approximately 150 CRD-rats were housed. At selected intervals the ex-germfree rats were killed and 47 examined by gross and histopathologic methods for pulmonary and tracheal lesions. In the first 2 experiments individual samples of trachea and azygous lobe of the right lung were cultured for bacteria and Mycoplasma g2, In the third ex- periment only lung was cultured. Egperiment 1. Four, 3-week-old, germfree Notre Dame-Wistar rats were removed from the germfree environment and placed near the center of a rack of galvanized wire cages arranged so that CRD-rats were on all sides except infront. These ex-germfree rats were killed after 4 or 6-week intervals and examined for evidence of respiratory disease. Experiment 2. Twelve, 9-week-old rats of the Sprague-Dawley strain were removed from their isolator after it had become contaminated with bacteria. Designated as controls, 2 were killed and examined on removal from the isolator. To deter- mine speed of natural transmission and rate of development of the lesions of CRD, the remaining 10 rats were housed in plastic "shoebox" cages fitted with wire tops and placed within a radius of 5 to 6 feet from cages housing the CRD— rats. At intervals of l, 2, 4, 5 or 8 weeks, 2 of the ex— germfree rats were killed and examined for evidence of res- piratory disease. Experiment 3. Four litters of Notre Dame-Wistar rats with 9 young rats per litter and their dams were removed from a 48 contaminated isolator and exposed to CRD—rats by the same techniques used in Experiment 2. At weekly intervals for 9 successive weeks, 1 rat from each litter was killed and examined. Ages of the individual litters at the beginning of the experiment were 35, 19, 8 and 2 days, respectively. Each dam was removed when her litter had reached a weaning age of 21 days (oldest litter was weaned before the experi- ment started) and was not evaluated as part of the experi ment. Exposure by Aerosol Inoculation. In this series of 8 experi— ments, germfree of ex-germfree rats were exposed to aerosols of a cell-free filtrate of pneumonic tissue or of broth cultures of Mt pulmonis. The rats, type and amount of inoculum, and day that exposed rats died or were killed are summarized in Table 1. Experiment 4. A pair of adult germfree rats was transferred to a separate germfree isolator and mated. When their off— spring reached two days of age both young and adults were inoculated with a cell-free filtrate freshly prepared from a pooled suspension of pneumonic lungs from CRD-rats (see later sections for details of preparation and inoculation of the cell-free filtrate). This procedure was used as it was the most convenient and practical means of making very young germfree rats available for aerosol inoculation. In 49 addition, it permitted comparison of the experimentally produced disease in both young and adult members of a single family of germfree rats. Experiment 5. One adult female and 3 adult male germfree rats were transferred to a separate germfree isolator for this experiment. The number and sex of the rats used in this experiment were determined by their availability at the time the experiment was conducted. The inoculum was prepared from an isolate of Mt_pulmonis designated as the Michigan/Jersey - 1969 strain (M/J-69). It was originally isolated from the pneumonic lungs of the adult female rat used in Experiment 4 (see preparation of Mt_pulmonis for inoculation). Experiment 6. The 8 male and 8 female ex-germfree rats of this experiment were housed, 4 per cage, according to sex in clear plastic "shoebox" cages fitted with stainless steel wire tops and filter caps. The cages, wood chips for litter, and water provided initially had been sterilized by auto- claving to establish a high level of sanitation. Sterilized litter and tap water were used for subsequent routine main— tenance procedures. The diet provided both initially and for maintenance had not been sterilized (see diet for con- ventional rats in the section on rations). The rats were placed in the cages immediately after removal from the germ- free isolator and were inoculated several hours later on the 50 same day. Designated as controls, the rats of one cage of 4 males and another cage of 4 females were exposed to an aerosol of sterile broth. The 8 rats in the two remaining cages were exposed by aerosol with the M/J-69 isolate used in Experiment 5. Ampules of the broth that had been frozen and stored at -70°C were thawed approximately 1 hour prior to inoculation. After the inoculations were completed, the cages with the controls and the infected rats were placed for the duration of the experiment at Opposite ends of a room at Barn 5. Experiment 7. Four adult female germfree rats were trans- ferred to a separate germfree isolator and inoculated with a subculture of the M/J-69 isolate. Procedures used were the same as in other experiments of this series in which germfree rats were inoculated with broth cultures of Mt_ pulmonis. Some additional details are included in Table 1. Experiment 8. Twelve adult germfree rats were inoculated with a broth culture of Mt pulmonis that had been isolated from the pneumonic lungs of a rat infected in Experiment 5. In addition, 4 young rats were born to l of the adult females 6 days after inoculation and were included in this experiment. Other details are included in Table 1. Experiment 9. Six adult and a litter of 8, 2-week-old germ- free rats were inoculated with a broth culture of Mt_pulmonis 51 that had been isolated from the pneumonic lungs of a rat from Experiment 6. The adults were killed and examined after they had developed signs of respiratory disease. The young rats were to be maintained until adulthood and mated to study the parent-to-offspring transmission of the infec— tion in subsequent generations. However, what appeared to be a nutritional deficiency developed in these and other germfree rats maintained in the isolators at Barn 5. Before the nutritional problem was brought under control by the addition of a multiple vitamin supplement to the drinking water, 5 of the 8 young rats had died and were cannibalized by their cage mates. The 3 surviving rats were maintained until they reached 32 weeks of age and were then killed and examined. Experiment 10. Two germfree isolators were prepared and 8 adult germfree rats were transferred into each. The rats in l isolator were inoculated with a freshly prepared subculture of the M/J—69 isolate. Those in the second isolator were inoculated with an equal amount of sterile broth. To obtain information on the early course of the infec— tion, some of the rats in this experiment were killed at short intervals after inocultion. Trachea and one tympanic cavity were cultured for Mt pulmonis in addition to samples of lung. Equal numbers of rats from each isolator were killed and examined at selected intervals following inocula— tion (see Table l). 52 Experiment 11. Two adult germfree rats were removed from a germfree isolator and were killed and examined. Five adult germfree rats that remained in the isolator were inocu- lated with a freshly prepared subculture of the M/J-69 isolate. The purpose of this experiment was to gain addi— tional data on the early course of the infection similar to some of the data obtained in Experiment 10. In addition to samples of lung and the contents of l tympanic cavity, samples of retrobulbar lacrymal gland (Harderian gland) were cultured for Mt pulmonis and examined histologically for inflammatory changes. Colony Controls. To confirm the absence of naturally occurr— ing CRD in the germfree rats the colony maintained at Barn 5, 7 aged females were removed from the main rearing isolator, killed, and examined for gross, histologic, and microbiolog- ical evidence of respiratory disease. These old breeders had been maintained in the main rearing isolator throughout the 18-month interval beginning prior to initiation of Experiment 4 and ending shortly after termination of Experi- ment 9. Rations A commercially prepared, pellected, autoclavable diet* specifically formulated for germfree rats and mice was used. *Mouse/Rat Diet (Autoclavable) (L-485). Teklad Inc., Monmouth, Ill. 53 .amc any we .maouucou mcoHou# coaumHsoDmH mcflsoHHOM mmmp m anon one; umcu mumm«*« .Oonm um vocabsocfl mum3 mflcoEHsm .2 mo mmusuaso cuoun Hm>umucfl mmumoflch«« .mumuuaflm mmHMIHHmoa ANVo I II mmumsumo N I om N ANVmHQm: v .umImv mmumfiumo m N om m HH Avvmm.0N.mH.b.v m mafluwum mwumeumw m m ma m Amvov.mN.0N.mH.h.v m .umINb mmumeumo m m ma m oa Anvo I II mmumEhmo n I mm b #00 AmvmmN mmumEhmw N H N m Amvna v .umINn mmnmeumo m m NH m m Avvov I II owHwEumo N N «««o v leewa.lmemfl.lmoma v .umuma magmaumu m m «a m Avvnm v .umINn mmuwsumu v I NH v s AvVHNH.ANVNm.ma.NH N waflumum mmumEMmmem w v m m AvVHNH.lmv~m.ma.~H N .umumq mmumeummuxm e v m m o Hm.ANv0N.mH m ;«.umImv commence H m ma v m AvvoaN.ANvmm.vm.ANvmv N «who mmumeumo v m a m mv.mm mmumeumu H H ma N v AHA we mumu .ozv AHEV buoum mmumeummem m z Amxsv .oz umbEdz been no omaaflx mumm noumHOmH no no xwm mod ucmEflummxm coflumHsoocflumom mama .I\u::0&¢ meme mmumEumo EsHsoocH mumm mucmeflummxo enamomxm HOmoumm CH com: cmfimmp HmucmEHdexw mo mnmEEsm .H manna 54 Three-pound quantities of the diet were placed in cloth sacks and autoclaved in a stainless steel sterilizing cylin- der. A 28-inch vacuum was maintained for 10 minutes and then steam was admitted and the temperature held at 122°C for 40 minutes. The diet was autoclaved in cylinders separate from any other materials to insure proper sterili- ation. This diet had been used successfully at Notre Dame University and was reported to be completely adequate for rats (Kellogg and Wostmann, 1969). Rats housed in conven- tional quarters were supplied a commercially prepared, pelleted ration* formulated for conventional rats and mice. Germfree Techniques Germfree rats were maintained in 24" x 24" x 60" flexi- ble plastic isolator units (Trexler, P. C., 1959) using standard germfree techniques (Sacquet, 1968). Within the isolators, the rats were housed in autoclavable, plastic "shoebox" cages fitted with stainless steel wire covers. The isolators were entered approximately once weekly to replenish food, water and litter and to remove waste material. The diet was autoclaved in cylinders separate from any other materials to insure proper autoclaving. Distilled water was autoclaved at 122°C for 1.5 hours in 1 gallon Square Pak Flasks** fitted with disposable self-sealing caps**. Once sterile, the flasks were stored in a clean *Peerless Laboratory Diet, A. K. Zinn and Co., Battle Creek, MI. **American Sterilizer Company, Erie, Pennsylvania. 55 area free of oil or grease and protected from accumulations of dust and dirt. As needed, the flasks were placed in the entry lock of the isolator and sterilized with 2% peracetic acid. The litter consisted of finely ground wood chips which were packaged in brown paper bags (S-pound size) and preautoclaved for a minimum of 1.5 hours. The prevacuum step as described earlier under rations was essential in the procedure for autoclaving litter. The preautoclaved pack- ages of litter were stored in a clean dry place and re- autoclaved for 1.5 hours in a transfer cylinder prior to use. Isolators were tested for sterility on a weekly schedule according to the method of Fuller (1968). Occasion- ally, the pressure of other duties forced deviations from this schedule but only rarely did the interval between sterility tests exceed 10 days. Generous mixed samples of water, litter and feces were collected in screw-top tubes, removed from the isolator, and taken to the Clinical Micro- biology Laboratory for culture and microscopic examination. In addition, the intestinal contents of germfree rats were cultured at necropsy to further assess their microbial status. Preparation of Cell-Free Filtrate Pneumonic lungs were collected aseptically from 5 CRD- rats that had been maintained in Room 345 Giltner Hall for a period of 6 months. The composite sample was minced and ground in a Ten Broeck tissue grinder using brain-heart 56 infusion broth to achieve a final dilution of 1:10. The suspension was centrifuged at 10,000 r.p.m. for 30 minutes, and the supernatant filtered under reduced pressure through a membrane filter* of 0.45 u average pore diameter. Thio- glycolate broth and blood agar plates were inoculated with some of the filtrate to test for bacterial contamination. Mycoplasma culture media were also inoculated. The remain- ing filtrate was transferred in 1 ml. amounts to glass ampules which were subsequently sealed by direct flame. Two amuples were used immediately to inoculate a group of germ- free rats (Experiment 4) and the others were frozen in a mixture of dry ice and alcohol and stored at -70°C. Technigues for Isolation and Growth of M. pulmonis Mggtg. Media for isolation and propagation of Mt_pulmonis were prepared from commercially available, dehydrated pro- ducts according to the directions provided by the supplier. Agar** and broth*** media were enriched by adding 30% of a commercial supplement**** prior to use. The supplement provided a final concentration of 20% horse serum and 10% yeast extract in both media. In addition, appropriate levels of thallium acetate and penicillin to inhibit bacter- ial growth were provided by the supplement. *Metricel Filter, Gelman Co., Ann Arbor, MI. **Bacto—PPLO Agar, Difco Laboratories, Detroit, MI. ***Bacto-PPLO Enrichment Broth, Difco Laboratories, Detroit, MI. ****Bacto—Mycop1asma Supplement S, Difco Laboratories, Detroit, MI. 57 Isolation of M. pulmonis from lung. A specimen of lung was removed aseptically from the carcass and placed in a sterile container. It was finely minced with sterile scissors, transferred to 5 m1. of broth medium in a screw-cap tube, and incubated at 37°C for 72 hours. At that time 0.1 ml. of the broth was transferred to the surface of an agar plate and another 0.1 ml. to a second tube containing fresh broth. Following incubation at 37°C for 72 hours, the plates were examined under the low-power objective of a microscope for colonies of Mt pulmonis. Specimens were considered negative if colonies were not observed on 3 successive passages. The specimen of lung was selected from a grossly abnormal portion of the lungs when possible. In the absence of grossly discernible lesions, the sample was taken from the apical portion of either the right or left lung. The latter sites were selected since these appeared to be the areas most consistently involved in lungs with gross lesions. In the natural transmission experiments, the azygous or un- paired lobe of the right lung was cultured. Isolation of M. pulmonis from trachea. A segment of the trachea measuring approximately 0.5 cm. in length was asep- tically removed midway between the larynx and the thoracic inlet and placed intact in the broth medium. Subsequent procedures were as described above for the lungs. 58 Isolation of M. pulmonis from tympanic cavity. The skin was reflected from the side of the head and the external ear was severed slightly lateral to the tympanum. Brain-heart in— fusion (BHI) broth was introduced through the tympanic membrane using a sterile syringe and needle and the contents of the cavity aspirated back into the syringe. A drop or 2 of the aspirate was transferred to a tube of the broth medium and evaluated as described above for the lungs. Only 1 tympanic cavity was cultured from each rat where this parameter was evaluated. Identification of M.gpulmonis Correct identification of the pathogen used in this research was essential in determining the etiology of the disease. The identification of an unknown mycoplasma is based on a variety of factors such as source of the organism, growth characteristics and colonial morphology, biochemical tests, response to inhibitors, and immunological properties. The growth inhibition test described by Clyde (1964) was used to identify the organism used for the experimental transmissions. Anti-Mt_pulmonis, -Mt arthritidis, and 'fl; pneumoniae sera purchased from a commercial laboratory* were used for the growth inhibition test. In this test, the organism is identified on the basis of an area of inhibited growth surrounding a paper disc impregnated with specific antiserum. Another immunological method for identification *Microbiological Associates, Bethesda, MD. 59 of mycoplasma was described by Del Guidice gt gt. (1967) in which they identified the organism by use of immunofluores- cent staining of colonies on agar. Preparation of M. pulmonis for Inoculation The general procedure was to incubate a pure culture of Mt pulmonis in mycoplasma broth medium, aerobically, at 37°C for 48 to 72 hours and to use the freshly prepared culture as the inoculum for an experimental transmission. Since there were occasional deviations from this general proce- dure and because proper preparation of the inoculum appears to have been particularly important for the successful com- pletion of this research, the details of preparation will be given. Caution was exercised throughout to avoid subject- ing the isolates of Mt pulmonis to potentially adverse conditions and thereby possibly causing a partial or total loss of pathogenicity. In particular, prolonged incubation without subculturing and excessive numbers of 12.23332 passages were avoided. Initially, Mt pulmonis was isolated in pure culture from the pneumonic lungs of an adult germfree rat that had been inoculated with cell-free filtrate (Experiment 4). Designated M/J-69, the isolate was passed 4 times in broth medium at 72-hour intervals. For the fifth passage, 0.1 ml. of fourth passage broth was transferred to 35 ml. of fresh broth medium. Following incubation at 37°C for 48 hours, 1.5-m1 volumes of the culture were flame-sealed in 60 sterile glass ampules. The ampules were frozen by swirling in a mixture of dry ice and alcohol and stored at -70°C. A blood agar plate and tubes of BHI and thioglycolate broths were inoculated with the 48-hour culture to test for bacter- ial contaminants. For subsequent transmissions, frozen ampules of M/J-69 were thawed and used directly as the infectious inoculum. Alternatively, material from the ampules was used to inocu- late fresh broth medium which was harvested after 48 or 72 hours of incubation and this freshly prepared broth was used as the inoculum. For still other transmissions, Mt_pulmonis was re-isolated from the pneumonic lungs of a rat infected with M/J-69 in an earlier trial and an inoculum was prepared using the new isolate. At each stage in the preparation of the various iso- 1ates for use as inocula, plates of agar medium were inocu- lated to be certain the organism remained viable. Similarly, bacterial culture media were inoculated to test for bacterial contamination. Passage of Infectious Inocula into Isolators Infectious inocula were placed in flame-sealed glass ampules for passage into the isolators. The ampules were washed with 70% alcohol to remove any dirt or grease accumu- lated from handling, placed in the entry lock of the isola- tor, sprayed with fresh 2% peracetic acid, and after 30 61 minutes taken into the isolator. A period of 30 to 45 minutes was allowed for expulsion of any peracetic acid that had been introduced into the isolator with the ampules. Lutsky and Organick (1968) emphasized that use of properly sealed glass ampules is essential to protect viable organ- isms from the peracetic acid in the entry lock of the iso- lator. Aerosol Inoculation of Rats The amount and type of inoculum used in each trial are given in Table l. A nebulizer* with attached rubber bulb was used to direct an aerosol of the inoculum toward the rats being inoculated. This nebulizer was reported to produce aerosols in which the majority of the particles are l to 5 u in diameter (Kundsin, 1968). Laurenzi and Guarneri (1966) reported that drOplets 3 u in size or smaller pene- trated the entire system of respiratory passages and entered the alveoli. Jericho (1968) suggested that use of aerosols to experimentally infect the lungs was "mandatory" because only with the aerosol method were all of the body's respira- tory defense mechanisms by-passed. In order to prolong the period during which the rats were exposed to the aerosol and to produce a higher concen— tration of the droplets in the air, the flow of air into the isolator was interrupted prior to starting the inoculation *Model 640 Nebulizer, The Devilbiss Co., Toledo, Ohio. 62 and re-established 30 minutes following completion of the aerosol procedure. Manual dispersal of the inoculum usually required 30 to 45 minutes, thus the flow of air to the isolator was halted for an interval of 60 to 75 minutes. Ex-germfree rats were inoculated through a hole cut in the fiberglass filter caps attached to the cages. Following completion of the aerosol procedure, the hole in the filter cap was sealed with plastic tape. Gross and Microscppic Examination of Tissues Rats were killed with sodium pentobarbital administered by intraperitoneal injection. Immediately after death, the trachea and lungs were exposed and specimens of lung and trachea were collected aseptically for culture. The trachea was not cultured in all cases. The skin was reflected from the head, and material for culture was collected from the tympanic cavity. Larynx, trachea and lungs were removed intact and adequate fixative was infused into the trachea to slightly distend the lungs. Tissues were fixed in 10% buffered for- malin except for a small number of selected specimens in which Zenker's fixative was used. The head was excised at the atlanto-occipital joint and placed in formalin after the skin had been removed. Additional tissues which usually included liver and kidney were collected when their gross appearance suggested the pressence of lesions. 63 For histologic examination, transverse sections were made of the trachea and larynx, and at least 1 section was made from each lobe of the lungs. After removal of the lower jaw, the fixed head was soaked for 7 or 8 days in formic acid-sodium citrate decalcifying solution. A transverse nasal section was made at a point midway between the exter— nal nares and the medial canthi, and a transverse section of the middle ears was made through the center of the external auditory canals. All tissues were dehydrated in ethyl alcohol, cleared in xylene, and embedded in paraffin. Decal— cified tissues were washed in tap water, re-fixed in 10% buffered formalin and then processed the same as the other tissues. Sections were cut at 6 u and stained with hema- toxylin and eosin. The Giemsa stain for mycoplasma, the Gram stain for bacteria and Gomori's trichrome stain for collagen and smooth muscle were applied as indicated. The Manual gt_Histologic Stainigg Methods of the Armed Forces Institute of Pathology, Washington, D. C. (Luna, 1968) was the source of the procedures used for fixing, decalcifying, processing and staining tissues. RESULTS The general results of each experiment are reported according to the order in which the experiments appeared in the materials and methods section. Data of each experiment are included in either Table 2 or Table 3. Observations of clinical signs, gross pathology, and histopathology were similar in each experiment and are therefore summarized in separate sections. Special note is made of particularly typical or atypical results. Experimental Infections The exposures were conducted in 2 separate series of experiments. In the first series, ex-germfree rats were exposed to rats affected with naturally acquired CRD. In the second series, germfree and ex-germfree rats were inoculated with aerosols of a cell-free filtrate or broth cultures of Mt_pulmonis. Exposure to Natural Disease. A total of 50 ex-germfree rats was eXposed to CRD-rats for intervals of l to 9 weeks. Histologic lesions were observed in the lungs from 3 rats; however, grossly visible lesions of pneumonia were observed in only 2 rats (Table 2). Mycoplasma gp, were isolated from the lower respiratory tract of 6 rats and a non-patho- genic micrococcus from 1 rat. 64 65 Experiment 1. Mycoplasma gp, was isolated from the lungs and trachea of all 4 rats (Table 2). Micrococcus gp. (reported as a light growth) was isolated from the lungs and trachea of l rat. There were no gross lesions in the lungs. However, all had microscopic lesions of the lungs and trachea. Tra- cheal lesions consisted of epithelial hyperplasia, increased mucus production, epithelial and subepithelial infiltration of inflammatory cells, and small necrotic foci in the epithe- lial layer. Mucopurulent exudate was scattered within the lumen. NeutrOphils predominated the cellular infiltrate within the epithelial layer, whereas lymphoid elements pre- dominated in the cellular changes beneath the epithelium. Microscopic lesions in the lungs consisted of mild peri- bronchial lymphoid hyperplasia and moderate bronchial epithe- lial hyperplasia. Alveolar consolidation was not observed in any of the lungs. The lesions were slightly more extensive in rats killed after 6 weeks than in those killed after 4 weeks. Experiment 2. Mycoplasma gp, was isolated from trachea and lung of l rat (Table 2). No bacteria were isolated from samples of trachea and lung. The lungs of 2 rats, includ- ing the one from which Mycoplasma gp_was isolated, had dark red areas of consolidation. The bronchi leading to the most extensively consolidated lobes were distended with thick, yellowish, muc0purulent exudate. 66 .UmcHmew uoz*«« .maumuomn now w>Hummmc mums mmHmEMm nmcuo Had .Hx.oz ucmEHHmmxm ca you H «0 mmcsa new wonomuu we» Eoum bmumHOmH was amm msooooonoflz 4 .mEHu meow um mflwmuomn How Umusuaso mums mmcsa bcm mmnomuewg .bmmomxm cmc3 mmumucH mumm mcoflqu mo mocmsqmum w muflm emu pmuflsqom waamusumc cues pmuommmm mumu Hmcoflucm>coo ou mum“ mmuwfihmmem Nm mo musmomxm Eoum sump mo xumEEsm .N magma 67 There were histologic lesions in the lungs of all rats in this experiment including the 2 rats that had been killed within an hour after removal from the isolator. In the 2 control rats the pulmonary lesions were small focal accumu- lations of macrophages, lymphocytes and eosinophils located in and around small bronchioles and in alveoli. Occasion- ally, small collections of eosinophils or lymphocytes were observed at the periphery of blood vessels and bronchi. The lymphoid tissue normally seen in the region of the large bronchi appeared to be slightly increased in amount. No inflammatory changes were observed in sections of trachea from either control rat. The histologic lesions in the lungs of rats that had been eXposed but that did not develop gross lesions were similar in appearance and distribution to lesions in the lungs of control rats, although the size of the individual foci was slightly larger in the exposed rats. An inflamma- tory reaction limited to approximately one-fourth the circumference of the trachea and characterized by subepithe— lial infiltration of lymphocytes and thickening of the over- lying epithelium was observed in 1 rat. In the lungs of the 2 rats with grossly apparent pneu- monia, the microsc0pic alterations were typical of these seen in the CRD syndrome. Specifically, the major changes were marked lymphoid hyperplasia in and around the walls of bronchi and blood vessels, dilated bronchi and bronchioles filled with mucopurulent exudate and necrotic debris, 68 variable areas of hyperplasia, atrOphy, and necrosis of the bronchial epithelium, areas of atelectasis, emphysema, and mucopurulent consolidation of the alveolar tissue, and zones of proliferating fibroblasts and formation of collagen fibers. The inflammatory lesions in the tracheas of these rats were quite similar in appearance to the lesions described earlier in rats from the first trial. These included hyper— plasia and necrosis of the epithelium, and infiltration of inflammatory cells into the wall of the trachea. Experiment 3. Cultural examination of the lungs of 36 young rats used in this experiment resulted in isolation of Mycoplasma gp, from only 1 rat (Table 2). No bacteria were isolated from the lungs. On gross examination, no lesions were seen in the lungs; however, histologic examination revealed small focal collec- tions of macrophages, lymphocytes, and cellular debris in the alveolar tissue of the lung from 5 rats. In the lungs of 2 other rats, the peribronchial lymphoid tissue was in- creased in amount and the bronchial epithelium was increased in height. One of the latter rats was the source of Myggf plasma gp, isolated during microbiologic examination. Tracheitis, characterized by subepithelial infiltration of macrophages and lymphocytes and epithelial hyperplasia was observed in sections of trachea of 4 rats. Two of the 4 were among those that had pulmonary lesions. Like the 69 pulmonary lesions, the inflammatory changes in the tracheas were not extensive. In each case less than one-half the circumference of the trachea was inflamed. Exposure by Aerosol Inoculation. A total of 65 germfree and ex-germfree rats were exposed to aerosols of a cell-free filtrate prepared from pneumonic tissue or to isolates of Mt pulmonis. Gross lesions of pneumonia were observed in 34 rats and histologic lesions in 36 rats. Pneumonia was observed as early as 13 days following exposure (Table 3). The incidence of inflammatory changes was higher in sections of trachea and the tympanic cavity than in the lungs. On microbiologic examination, Mt_pulmonis was isolated from the lungs of 40 rats. When the trachea and tympanic cavity were cultured, Mt pulmonis was isolated from a higher percentage of rats than when only lung was cultured. In 1 experiment, none of the rats inoculated with a broth culture of Mt V pulmonis developed gross or histologic lesions of pneumonia and Mt_pulmonis was not recovered from their lungs. Experiment 4. Clinical sings of respiratory distress developed first in the adult male at 2 weeks after inocula- tion with the cell-free filtrate and shortly thereafter in the adult female. Clinical signs of illness were no observed in the young rats. On microbiologic examination, Mycoplasma gp, was isolated from the lungs of the adults and 3 of the 9 young rats (Table 3). 7O .Oonm um UmumnsocH mums mHCOEasm .EsHeme shown mHHumume 4w mo mwusuHso buoub Hm>uwucH mmumoHUcH««4« . UGCHEMvnw #OZi a. s .mHMHUHHm mmHMIHHwO«« .coHumHsoocH wo mEHp um mumn «0 mm«« mz mz N\o mz N\o N\o N\o m2 II om N N\H ¢\H m\m H\o m\N m\m m\v mz .HmIm¢ om m Ha mz m\o m\o m\o m\o m\o m\o m\o 2mm ma m mz m\m m\m >\v m\m m\m w\m m\m .HmINh ma w oa N\o N\N me m\N N\N mz N\N mz .umINN N m m\N m\m m\¢ m\v m\v mz w\v mz .HmINh NH m m mz mz v\o «\o «\o mz «\o mz II o v mz mz NH\HH NH\HH NH\HH mz NH\0H mz .umINh «a NH m mz mz «\o «\o «\o mz ¢\o mz .umINn NH e N mz mz m\o m\o m\o mz m\o mz *zmm m m mz mz N\e m\m m\v mz m\m mz .umume m m o «\N «\v v\v «\v v\q mz v\v mz*««mumIm¢ ma v m mz mz N\H m\m m\o mz mxm mz emu H m m2 m2 m\N N\N N\N m2 N\N «atmz «ammo ma N e muH>mu wuH>mu mmcsq mono omega (NuH>mO mmcsq mono EDA «Amx3v .02 .oz Hommz becomewe Immm MmsmmE>B IMHB IsoocH mod .Hmmxm mconwH oemoHoumHm mconmH whoHumHOmH choeHsm, mo mumm mmouo .2 mo wocmsqwum bum mama mcoflqu mo mwcmnmwum can muHm muwm mHGOEHom mEmem00wz mo mwusuHso cuoub no mmcsa awn oHcoEsmcm mo mumuuaflm mmHMIHHmo spas mumu mmuweummeo 0cm wmumEumm mo :oHumHsoocH HOmoumm Eoum name no mumfifidm .m meme 71 At necropsy, gross lesions of pneumonia were observed in both adults but not in the young rats. Histologic lesions were found in the lungs of 1 young rat in addition to those in the pneumonic lungs of the adult rats. The pulmonary lesions observed in the young rat were small foci of consolidation unaccompanied by alterations in other portions of the lungs. Specifically, there was no evidence of peribronchial lymphoid hyperplasia or of bronchiectasis in this rat. The lesions in the lungs of the adult rats were typical of those produced throughout this series of eXperiments and will be described in a later section. Tracheitis, characterized by epithelial hyperplasia and subepithelial infiltrations of lymphocytes, macrophages and occasionally a few neutrophils, was observed in the 2 adult rats and in 6 of the young rats. The epithelium was greatly thickened and the inflammatory changes were well developed in the tracheas of the adult rats. With the exception of the young rat that had microscopic lesions in the lungs, the lesions in the tracheas of young rats were never as extensive as those in the older rats. The isolate of Mycoplasma gp, from the lungs of the adult female rat designated as M/J-69 was identified as Mycgplasma pulmonis by means of a growth-inhibition test. It was propagated in broth and used to inoculate the rats in several of the following experiments. 72 Experiment 5. This was the first experiment in which the rats were inoculated with pure cultures of Mt_pulmonis. The rats were killed between days 15 and 31 postinocula- tion and after the onset in each case of clinically severe respiratory distress. The progress of clinical signs in the 3 males preceded the development of similar signs in the female. The results of microbiologic and pathologic examinations were essentially the same for each rat (Table 3). Lesions included gross consolidation and bronchiectasis in the lungs, tracheitis, and bilateral otitis media. Transverse sections through the nasal cavities revealed rhinitis in 2 of the 4 rats. Microbiologic examinations of consolidated portions of the lungs resulted in isolation of Mt_pulmonis from all 4 rats. No bacteria were isolated. Experiment 6. In this experiment 8 male and 8 female, 5-week old, ex-germfree rats were placed in 4 cages with 4 males or 4 females in a cage. The cages were fitted with filter-tops made of compressed fiberglass. The rats in 1 cage of males and another of females were inoculated with a broth culture of Mt pulmonis. Rats in the remaining 2 cages were inocu— lated with sterile broth. Clinical signs of respiratory disease had not develoPed 2 weeks after inoculation when 1 rat from each cage was killed and examined. Cultural examination of the lungs re- sulted in isolation of Mt_pu1monis from 1 rat in the infected 73 group (Table 3). Gross and histopathologic lesions were not observed in the lungs and tracheas. Another group of 4 rats was killed 82 days postinocu— lation and the remaining 8 rats were killed 4 months postin- oculation. Four of the 6 inoculated rats had gross lesions of pneumonia, Mt pulmonis was isolated from their lungs, and tracheitis was observed histologically. Tracheitis was also observed in l of the other 2 rats. Comparable examina- tions of the control rats were negative in each case. Experiment 7. In this experiment, 4 adult female germfree rats were inoculated with a broth culture of Mt_pulmonis and killed on Day 57 postinoculation. Cultural examinations of their lungs were negative for Mt pulmonis. No gross or histopathologic lesions were observed in their lungs and trachea (Table 3). Experiment 8. Twelve adult germfree rats were inoculated with a broth culture of E; pulmonis for this experiment. On Day 6 postinoculation, one of the inoculated females gave birth and 4 young rats from the litter were included in the experiment. The adult rats were killed or died between Day 13 and Day 33 postinoculation. Gross lesions of pneumonia were present in 11 of the 12 adults and Mt_pu1monis was isolated from the pneumonic lungs of 10 (Table 3). An attempt to isolate Mt pulmonis from the lungs of a rat that had died 74 and was refrigerated overnight prior to collection of a sample of its lungs for culture was unsuccessful. As indi- cated above, 1 of the 12 adult rats did not have pneumonia at necropsy. On histologic examination no lesions were observed in the lungs and trachea of that rat. Tracheitis was observed in the 11 rats that also had pneumonia. The 4 young rats were killed at 4 weeks of age. There were no gross or histologic lesions in the lungs and tra- cheas of the young rats and Mt pulmonis was not isolated from the lungs. Experiment 9. Four of 6 adult rats that were killed 16 days after inoculation had gross lesions of pneumonia (Table 3). Upon microbiologic examination of the lungs, Mt pulmonis was isolated from 4 of the rats. Histologic lesions were observed in lung, trachea and tympanic cavities of the 4 rats that were positive on culture for Mt pulmonis. In addition, histologic lesions of rhinitis were observed in 2 of the latter rats. Otitis media was observed in l rat that was negative on culture for Mt pulmonis. Pneumonia and tracheitis were observed in 2 rats killed 32 weeks postinoculation and Mt_pu1monis was isolated from their lungs. The third rat killed at that time did not have pneumonia and Mt pulmonis was not isolated from its lungs; however, otitis media was observed histologically in this rat. The above 3 rats were 2 weeks of age at the time of 75 inoculation. A further history of these rats is given in the section on materials and methods. Experiment 10. Eight adult germfree rats were inoculated with a broth culture of Mt_pulmonis, and a comparable group in another isolator was inoculated with sterile broth. The rats from each group were killed between 4 and 44 days postinoculation. Samples from tympanic cavity, trachea and lung of each rat were cultured individually for Mt_pulmonis (Table 3). No microorganisms were isolated from the control rats. Cultures of tympanic cavity and trachea of 8 rats and of lung from 6 rats in the infected group resulted in iso— lation of Mt pulmonis. Lungs from a rat killed on Day 4 and a rat killed on Day 44 were negative for Mt_pulmonis. Microscopic lesions were present in sections of tympanic cavity from rats killed throughout the experiment. Lesions were not observed in lung and trachea of rats killed prior to Day 14 postinoculation. Experiment 11. Three rats were killed on Day 7 and another 2 rats on Day 14 postinoculation and a retrobulbar lacrymal gland from each was cultured for Mt pulmonis. The contra- lateral gland was examined histologically. The cultures were negative for Mt_pulmonis, and no microscopic lesions were observed in the lacrymal glands. Myc0plasma pulmonis was isolated from the tympanic cavities of all 5 and from the lungs of 4 of these rats (Table 3). Two control rats 76 that were killed and examined prior to inoculation were negative for Mt_pulmonis from all 3 sites, and there were no gross or microsc0pic lesions in lacrymal gland, tympanic cavity, trachea or lungs. Neoplasms were an incidental finding in 3 of the aged germfree rats used in this experiment. There were adenomas in the mammary glands of 2 rats, and 1 rat had a thymoma in the mediastinum and a fibroma located on the external ear. Colony Controls. Samples of lungs from each of the 7 rats were cultured for Mycoplasma sp. and bacteria. No micro— organisms were isolated from any of the specimens. Upon gross examination, numerous small white spots measuring up to 0.1 cm. in diameter were visible at the surfaces of the lungs of 4 rats. Microscopically, there were focal accumulations of large foamy macrophages mixed, in some cases, with faintly staining amorphous debris. These foci were randomly distributed throughout the alveolar tissue of the lungs. The microscopic appearance of a repre- sentative lesion is illustrated in Figures 1 and 2. Some of the foci were situated just beneath the visceral pleura and were undoubtedly the white spots seen on gross examina- tion. Occasionally, 2 or more adjacent foci had coalesced to form relatively extensive accumulations. The alveolar and bronchiolar tissues within the involved areas did not appear to be affected. Figure l. Vacuolar macrophages and amorphous debris in alveoli of lB-months-old germfree rat. H & E stain. x 130. Figure 2. Higher magnification of an area from Figure 1. Note the apparent lack of involvement of bronchial and alveolar tissues. H & E stain. x 325. 78 No microscopic evidence of inflammatory reactions or other abnormalities were observed in other portions of the lungs or in sections of trachea, middle ear, and nasal turbinates. Figures 1, 2, 5, 9, l4, 19, 20, 25, 28, 31, 33, 37 and 39 are representative of the tissues from these rats. Clinical Signs The experimental disease was usually inapparent in rats that did not have gross lesions of pneumonia on post- mortem examination. However, even when clinical signs were present, it was seldom possible to correlate the clinical appearance of a rat with the severity of his pulmonary lesions. On the other hand, rats with extensive consolida- tion of the lungs did not always have obvious clinical signs. As can be seen by comparing a healthy adult male germfree rat (Figure 3) with an adult male rat 3 weeks following inoculation with an aerosol of Mt_pulmonis (Figure 4), the outward appearance of infected rats was not markedly altered. Although a reduced growth rate and dyspnea were readily evident in the rat with CRD, only slight roughness of coat was clearly discernible from the photograph. At necropsy shortly after the photograph was made the lungs were exten- sively consolidated (Figure 6). The initial clinical signs usually were rough hair coat, lethargy and increased respiratory rate. Later and more serious signs were dyspnea, anorexia, rapid loss of weight, and humped posture. Terminal signs included mouth breathing 79 Figure 3. Healthy adult male germfree rat Note smooth glistening coat. Figure 4. Adult male rat three weeks following inoculation with aerosol of broth culture of E; pulmonis. Although reduced weight gain, emaciation and dyspnea were readily evident in this rat, only slight roughness of coat is clearly discernible in photograph. 80 with the neck extended and intermittent episodes of violent physical activity, apparently due to transient respiratory obstructions. The clinical picture described above was observed in adult rats beginning, at the earliest, 7 to 10 days after inoculation. Rats that were infected at less than 3 weeks of age did not develop rapidly progressive respiratory disease. On one occasion clinical signs were observed in rats that had been inoculated at an early age. Two of 3 rats that had been inoculated at 2 weeks of age deve10ped signs shortly before they were killed at 32 weeks of age. Both of these rats had pneumonia at postmortem examination. In adult rats, the incidence of rapidly progressive disease with the development of obvious pneumonia was high. However, while some rats within a cage were near death, others in the same cage remained essentially unaffected. For example, in Experiments 8 and 9, 18 adult rats were killed between 2 and 3 weeks after inoculation, gross pneu- monia had developed in 15, whereas the lungs of 3 were free of gross and microscopic lesions. Pathology The macroscopic and microsc0pic lesions observed in the rats from each of the aerosol inoculation experiments were essentially the same. Any distinguishing features between the lesions of rats from different experiments were readily explained by the length and intensity of the 81 infection. In other words, the differences were quantita- tive rather than qualitative and often varied as much among the rats of a single experiment as they did between experi- ments. The summaries of gross and microscopic lesions given below pertain primarily to the aerosol inoculation experiments (Experiments 4 - 11). The very limited lesions observed in rats exposed to natural disease (Experiments 1 - 3) are described with the general results of those experi— ments. Gross Patholog . Emaciation was the most common sign of disease observed on external examination of the carcasses at necropsy. However, the frequency of this observation was undobutedly influenced by the practice of awaiting the onset of severe clinical signs before the rats were killed and examined. In addition, there were scanty accumulations of brown or red-brown exudate on the muzzles of a few rats. On examination of the thoracic viscera, a characteristic and rather constant observation was compensatory emphysema of the unconsolidated portions of lungs from rats that had pneumonia (Figure 6). With loss of elasticity, the lungs did not deflate and collapse as normally occurs when the negative pressure within the thoracic cavity is released. The magnitude of the emphysema varied from one rat to another, but in general was greatest in cases with extensive and longstanding pulmonary consolidation. P U i g. U 2 Figure 5. Hilar view of individual lobes of lungs from germfree rat in Figure 3. Lungs were infused with saline solution via the trachea prior to dissection. The lobes are labeled; (A) apical, (B) cardiac, (C) diaphragmatic, (D) azygous lobes of right lung and (E) left lung. Figure 6. Pneumonic lungs of rat 4 weeks follow— ing aerosol inoculation with g; pulmonis. Notice total consolidation of right apical lobe and left lung, and hypertrophied right cardiac lobe. 83 The macroscopic pulmonary lesions were consolidation and bronchiectasis. Consolidation usually began in the apical portion of the lungs as was evident from cases of relatively short duration (Figures 7 and 8). At this stage, the areas of consolidation were dark red in color, mildly indurated and slightly depressed at the surface. Later the consolidation had usually spread to include more of the pulmonary tissue and appeared mottled with dark red areas interspersed with areas of yellow-gray coloration (Figure 6). Often entire lobes became consolidated and frequently, as illustrated in Figure 6, the left lung was affected in this manner. Regardless of the stage of examination, less obvious foci of consolidation were usually present in other lobes of the lungs, some being visible on examination of the cut surface transected lobes. However, observations of consolidation in all lobes of the lungs from a given rat were uncommon. Bronchiectasis, characterized by markedly distended bronchi containing yellowish, semi-fluid, mucopurulent exudate, preceded or accompanied the onset of consolidation. As illustrated in Figure 8, bronchiectatic bronchi were commonly seen in unconsolidated lobes indicating that bronchiectasis preceded consolidation. Commonly, circum- scribed, elevated, yellow-colored areas having the appearance of abscesses were seen on the surface of the lungs. These focal lesions represented markedly dilated, pus-filled bronchi rather than true abscesses and will be discussed in 84 METRUC 1 Figure 7. Dorsal view of lungs 15 days following aerosol inoculation with g; ulmonis. Consolidation of apical region of left lung an apical lobe of right lung were typical distribution of the pulmonary lesions at this stage of the infection. Figure 8. Ventral view of lungs 20 days following aerosol inoculation with g; pulmonis. Note consolida- tion of apical and cardiac lobes of right lung and apical region of left lung. Also marked bronchiectasis, subpleural ecchymoses, and mucopurulent exudate drain— ing from trachea. 85 greater detail in the section on histopathology. In cases of longer duration, the bronchial exudate had changed from its earlier semi-fluid mucopurulent character to a semi-moist almost caseous consistency. Pleural adhesions and other indications of pleuritis were not observed in any of the experimentally infected rats. The small size of the trachea of the rat made gross determination of lesions difficult; thus emphasis was placed on its proper preservation for histologic examination. Nevertheless, in many cases, the lumen of the trachea con- tained variable quantities of mucopurulent exudate as illustrated in Figure 8. Other parts of the respiratory system were not routinely examined for macroscopic lesions, either because of emphasis on lesions that developed in the lungs, as was the case during the early experiments, or because it was more desirable to preserve the anatomical structures intact for subsequent histologic examination. In a few instances, the osseous tympanic cavity was opened and in each case the cavity contained creamy yellow purulent exudate. In other cases, on close examination after the external ear had been severed near its base, the tympanic membrane bulged outward and was distinctly yellow in color. HistOpathology. The distribution and general character of the bronchOpulmonary lesions observed in rats following aerosol inoculation with §;_pulmonis was represented by the 86 tissues in Figure 10 and 11. The dilated bronchi with greatly thickened walls were best appreciated when compared to the bronchi in comparable sections of normal lung (Figure 9). Although the term consolidation was used to describe the macroscopic appearance of lungs, such as that illus- trated in Figure 11, the microscopic appearance of the tissue indicated that atelectasis was the predominant alter- ation of the alveoli (Figures 12, 13 and 14). Atelectasis did not account entirely for the consolidation observed, as areas of true consolidation characterized by distention of alveoli with inflammatory exudate were observed in some lungs (Figures 15 and 16). These areas of true consolida- tion never involved large portions of an infected lung, and were often difficult or impossible to find in the cross section of an entire pulmonary lobe. Alterations of the alveoli appeared to result secondarily from the inflammatory changes occurring in and around the bronchi and bronchioles. Epithelial hyperplasia accompanied by increased production of mucus, peribronchial lymphoid hyperplasia, and peribronchial infiltration of lymphoid cells were observed initially in the large bronchi (Figure 17). Neutrophils invaded the hyperplastic bronchial epithe— lium where they were observed in small aggregates within microscopic foci of necrosis and also as solitary cells apparently in the process of migrating to the brinchial lumen (Figure 18). The large numbers of neutrophils within the bronchial epithelium probably were an indication of the 87 Figure 9. Sections through cardiac (A), and diaphragmatic (B) lobes of right lung and left lung (C) from an lB-months-old germfree rat. H&E stain. x 5. following aerosol inoculation with g; pulmonis. Com- pare to Figure 9 and note especially the diameter of the bronchi and thickened bronchial walls. H & E stain. x 5. Figure 11. Transverse sections through totally consolidated left lung of rat 16 days following aerosol inoculation with g; pulmonis. H a E stain. x 5. 9 mflsrw: -- i we Air-xix ' ' ms. ‘ k" ' a 3""1 . A «’1 KW Figure 12. Bronchiectasis and atelect sis in lung of rat 35 days following inoculation with M. ulmonis. Note extensive atelectasis (A), thickened Gal 0 dilated bronchus that lacks epithelial lining (B), bronchial exudate (C), large blood vessel (D), and pleural surface of lung (arrow). H & E stain. x 52. av ' _ . . w:- .,._-.v.«—- Figure 13. Pulmonary consolidation with prominent atelectasis and bronchiectasis from rat 15 days follow- ing aerosol inoculation with M. ulmonis. Note areas of atelectasis (A) and exudate—in dilated bronchi (B). H & E stain. x 52. Figure 14. Division of bronchi with adjacent alveolar tissue in lung from germfree rat. Note thick— ness of normal bronchial wall and bronchial epithelium (A), and lymphoid tissue in wall of bronchus (arrow). H 8 E stain. x 52. 90 \ V ... : " ~ A) Figure 15. Acute purulent pulmonary consolidation in rat 16 days following aerosol inoculation with g; pulmonis. H & E stain. x 52. Figure 16. Higher magnification of a portion of Figure 15. Note apparent streaming of neutrophils from lumen of bronchiole into alveoli. H & E stain. x 130. 91 source of the tremendous numbers of neutrophils that eventu- ally accumulated in the lumens of the bronchi. The peribronchial accumulations of lymphoid tissue appeared to arise from hyperplasia of the small aggregates of lymphoid cells normally present in or near the bronchial walls (Figures 19 and 20), and concurrently by diffuse infil- tration of lymphocytes into the peribronchial region (Figure 21). The amount of lymphoid tissue that eventually accumu- lated in and around the walls of large bronchi usually exceeded many times that which was observed surrounding the small bronchi (Figure 22). Concurrent with the inflammatory changes that were occurring in and around the bronchial walls c0pious quanti- ties of mucopurulent exudate accumulated within the lumens of the bronchi. During this time the walls of the bronchi apparently were weakened, and the bronchi became markedly dilated. The peribronchial apposition of lymphoid and other inflammatory cells and the bronchial dilatation were both space-occupying processes that proceeded at the expense of the adjacent alveoli, thus providing a direct explanation for at least some of the atelectasis described earlier (Figure 23). Dilated, pus-filled bronchi resembling abscesses were described in the section on gross pathology. Microscopically, these abscess-like formations were in each case identified as bronchi from scattered intact fragments of bronchial 92 Figure 17. Bronchial epithelial hyperplasia and atelectasis in lung of rat 16 days following aerosol inoculation with g; ulmonis. Note hyperplastic bronchial epithelium A) and atlectasis (B). H & E stain. x 52. 0' Figure 18. Hyperplastic bronchial epithelium lining bronchus of rat 18 days following aerosol inoculation with M. pulmonis. Note neutrophil in epithelial layer adjacent to luminal border (arrow). H & E stain. x 520. Figure 19. Lymphoid tissue in wall of bronchus from adult germfree rat. H & E stain. x 52. ’5‘. .n Figure 20. Lymphoid aggregate in wall of bronchus from adult germfree rat. Note uniform bronchial epi- thelial layer overlying lymphoid tissue. H & E stain. x 130. 94 Figure 21. Peribronchial lymphoid hyperplasia in rat 16 days following aerosol inoculation with M; pulmonis. H & E stain. x 52. xx .- s. I V ‘ a .2 “l .‘ . \ ‘ .\"o" x ‘t‘ «‘\M \‘ . V . ks I... \ " 3 T‘W' 4‘, -I ' ' . 3», w ... A ‘ : ‘ v ~ 4. Exudate-filled bronchus from rat 18 days Note Figure 22. following aerosol inoculation with M. ulmonis. purulent (A) and vacuolar macrophage—(B zones of bronchial exudate. Also note hypertrophic bronchial epithelium (C), and rather sparse peribronchial accumu- lation of lymphocytes (D). H & E stain. x 325. 95 epithelium. In some cases most of the epithelium was missing and the altered bronchial wall more closely resembled the pyogenic membrane of a true abscess (Figure 24) than the wall of a normal bronchus (Figure 25). In later stages of the inflammatory process, fibro— blasts proliferated and collagen fibers were formed in the peribronchial tissues and in the collapsed or consolidated portions of the lungs. Smaller bronchi and bronchioles were often ensnared by the maturing collagen fibers and greatly distorted as the fibers contracted (Figure 26). Although the proliferation of peribronchial connective tissue has been suggested as the mechanism by which bronchi become stabi- lized in the dilated state, it appeared that the bronchi were actually constricted, causing the lining epithelium to be thrown into deep folds. Neither papillary hyperplasia nor unequivocal squamous metaplasia of the bronchial epithelium was observed in the experimentally infected rats. Tracheitis, characterized by epithelial hyperplasia and subepithelial infiltrations of lymphocytes, macrophages and occasionally a few neutrOphils, was observed in all rats that had pulmonary lesions and in some rats that did not develop pneumonia (Figures 27, 29, and 30). The hyper- plastic epithelial growth resulted in the formation of deep glandular invaginations resembling in appearance the crypts of the large intestine. The thickness of the tracheal mucosa was increased several fold (compare Figures 27 and 28). The epithelial cells on the mucosal surface were flattened Figure 23. Bronchiectasis and partial atelectasis in rat 18 days following aerosol inoculation with g; ulmonis. Note markedly thickened bronchial wall and partial collapse of adjacent alveoli resulting from expansion of bronchus. H & E stain. x 52. ., \ . A ",1 _ o~‘?. . . ,. y. ‘ “, Figure 24. Intrabronchial abscessation in rat 19 days following aerosol inoculation with M. ulmonis. Note purulent (A), neutrophil (B), and vacuolar macro- phage (C), zones of the exudate, and remnants of bronchial wall (D). Also note collection of lymphoid cells (E). H & E stain. x 130. Figure 25. Small bronchi in lung of adult germ— free rat. H & E stain. x 130. t u- a.‘ o .. Figure 26. Distortion of small bronchi ensnared by maturing collagen fibers. Note irregularities in shape of lumens probably caused by constricting collagen fibers. H a E stain. x 130. - A-n . Transverse section of trachea from rat 15 days following aerosol inoculation with M. pulmonis. Note hyperplastic epithelium with deep invagihations (A), inflammatory cells in lamina propria and submucosa (B), submucosal glands (C), and thyroid gland (D). H & E stain. x 52. Figure 27. _ 1' Figure 28. Transverse section of trachea from adult germfree rat. Note height of epithelium (A), tracheal cartilage (B), and lumen of esophagus (C). H & E stain. x 130. Figure 29. Higher magnification of Figure 27. Note invaginations of hyperplastic epithelium (A), inflammatory cells in lumen of submucosal gland (B), in lamina propria (C), and in submucosa (D). H & E stain. x 130. Figure 30. Transverse section of trachea from rat 18 days following aerosol inoculation with M; pulmonis. Note hyperplastic mucosal epithelium (A), hyperactive mucus-secreting glands in mucosa (B), and in submucosa (C), and inflammatory cells in wall of trachea (D). H & E stain. x 130. 100 and resembled squamous cells. Hyperplasia of the sub- mucosal mucus-secreting glands located in the upper trachea and the mucus-forming cells in the mucosa facilitated increased production of mucus (Figure 30). Neutr0phils infiltrated the epithelium and could be found scattered individually throughout the mucosa or were present in small aggregates within foci of necrosis near the epithelial surface. Collections of neutrophils were often observed within the lumens of submucosal glands and in the Openings of the glandular invaginations into the mucosa. Mono- nuclear cells which were predominantly lymphocytes infiltra- ted all layers of the tracheal wall but were most abundant in the lamina propria and submucosa. The otitis media observed in rats following aerosol inoculation with M; pulmonis was characterized by the rapid accumulation of purulent exudate within the tympanic cavity. The normal tympanic cavity of the germfree rat was void of contents other than the auditory ossicles and related structures (Figure 31). The cavity was lined by simple squamous epithelium supported on a thin layer of loose connective tissue (Figure 33). Proliferating connective tissue extensively infiltrated the exudate within the tympanic cavities of infected rats and in some cases eventually filled by the connective tissue, the remainder being filled with purulent exudate (Figures 34 and 35). Within the connective tissue framework, small cystic spaces formed that were lined by simple cuboidal epithelium. These spaces were filled Figure 31. Transverse section slightly rostral to external auditory meatus of adult germfree rat. Note tympanic cavity (A) and osseous tympanic wall (arrow). H & E stain. x 5. Figure 32. Transverse section slightly rostral to external auditory meatus of rat 20 days following aerosol inoculation with M; pulmonis. Note organized inflammatory exudate in tympanic cavity (A), part of external auditory canal (B), and neural epiphysis (C). H & E stain. x 5. 102 “L4BE§\ \‘ ...‘ ; . l I K; ~ ’3 ' , ‘ . .-’.° ‘ - c ._ ' - . ‘H ' ‘ 1 -p - ‘O a .\3 r _, v ' ‘ ' r.“ U. - -\ - , '11.: -m _ m adult Figure 33. Wall of tympanic cavity fro germfree rat. Note tympanic lumen (A), simple squamous epithelium supported by thin layer of loose connective H&E tissue (B), and osseous tympanic wall (C). stain. x 130. 1,, ,Aggln,“ A , Figure 34. Ventrolateral region of tympanic cavity of rat 31 days following aerosol inoculation with M; pulmonis. Note purulent (A), neutrophil (B), and organized (C) zones of tympanic exudate. Also note cystic spaces (D), and blood vessels in organized regions of tympanic exudate (arrows). H & E stain. x 52. 103 with serous fluid and small to moderate numbers of inflammatory cells, and resembled newly formed lymphatic channels. The tympanic membrane of an affected ear became markedly thickened by proliferated epithelial cells and connective tissue. In one case the inflammatory reaction extended through the tympanic membrane into the external auditory meatus (Figure 36). Medial extension to the inner ear was not observed. In rats exposed to M; pulmonis infection there was a purulent rhinitis characterized by purulent exudate within the nasal passages, diffuse subepithelial infiltration of lymphocytes and alterations of the epithelial lining (Figures 38 and 40 in contrast to Figures 37 and 39). Microbiology The sites and frequencies of mycoplasma isolations are included in Tables 2 and 3. In each case cultures for bacteria were done in parallel with the cultures for myco- plasma. The bacterial data have not been included in these tables since bacteria (a nonpathogenic micrococcus) were isolated on only one occasion. Mycoplasma pulmonis was isolated from the lungs of all but one rat in which there was grossly detectable pneumonia. The rat from which the organism was not isolated had died spontaneously and was placed in a refrigerator overnight prior to culturing. In dealing with rats from the necropsy laboratory and from Figure 35. Higher magnification of area in Figure 34. Note simple cuboidal lining of cystic spaces (A), rich vascular network (B), zone of degenerating vacuo- lar macrophages (C), and vascular connections with osseous tympanic wall (arrows). H & E stain. x 130. .,.— . _I_ _, ~ ”,4 1" I . ‘3 . I), 1.. ‘_' {3,4 . ‘ I ‘ 7 . ‘x‘. '5’ '. ‘ ‘ ‘ g9 ‘~a Epfifft 7’ , 7.. é V‘ ... ' A ‘ i-I'A‘N - .r a. "Z .',-.., x." , . Figure 36. Outward extension of otitis media through tympanic membrane of same ear as Figures 34 and 35. Note external acoustic meatus (A), thickened tympanic membrane (B), parts of auditory ossicles (C), and inward growth of tympanic epithelium into tympanic exudate (D). H & E stain. x 52. Figure 37. Transverse section through nasal cavity of adult germfree rat. Note cartilaginous median nasal septum (A) and maxillary turbinate (B). H & E stain. x 52. Figure 38. Purulent rhinitis in rat 20 days following aerosol inoculation with M. pulmonis. Note cartilaginous median nasal septum (A), and purulent exudate in nasal cavity (B). H & E stain. x 52. Figure 39. Anterior ventrolateral nasal mucosa of adult germfree rat. Note distribution of the sub- epithelial lymphocytes. H & E stain. x 520. ‘i.J - .. 13;“ n H; w§*.-\4' Figure 40. Purulent rhinitis on floor of nasal cavity in rat 20 days following aerosol inoculation with M. pulmonis. Note purulent exudate in lumen (A), and flattened epithelial cells (B). H & E stain. x 520. 107 several unreported experiments, it was found that, in general, M;_pulmonis is difficult to recover from carcasses that have been refrigerated prior to culturing. These observations emphasize the necessity of promptly processing specimens when isolation of this organism is desired. Colonies of M;_pulmonis grown on agar medium were detected as early as 18 hours after the plates had been seeded with 48- to 72-hour broth cultures of the organism; however, the Optimum time to examine the plates was 72 hours after seeding. The typical appearance of the colonies of M; pulmonis is illustrated in Figures 41, 42, 43, and 44. 108 Figure 41. Colonies of M; pulmonis growing on agar medium. Incubation for 48 hours at 37C. Unfixed and unstained. x 52. Figure 42. Colonies of M. ulmonis growing on agar medium. Incubation for 72' ours at 37C. Unfixed and unstained. x 125. 109 agar medium. Incubation for 120 hours at 37C. Unfixed and unstained. x 130. Figure 44. Colonies of M; ulmonis on agar medium. Incubation for 120 hours at 37C. Unfixed but stained with amido black B to increase contrast. x 52. DISCUSSION In determining the cause of CRD the logical starting point was to use rats with the natural disease and to repro- duce it in susceptible rats of known microbial status. The CRD-rats were collected from several sources to increase the probability of including virulent strains of the patho— gen or pathogens in the diseased colony. These rats were housed under rather poor environmental conditions that provided suitable conditions for natural transmission of the disease. Cross transmission was desirable to maintain a high level of active respiratory disease among the CRD-rats and to increase the probability of adequate exposure when susceptible rats were introduced to investigate natural transmission of the disease. Exposure of Ex-Germfree Rats to Natural Disease The results from exposing 50 ex-germfree rats to the natural disease included isolation of Mycoplasma s2, from the lungs and trachea of 6 rats. In addition, 2 rats deve10ped gross pulmonary lesions typical of CRD, 11 had tracheitis, and in 23 there was a variety of mild microscopic pulmonary lesions. Evaluated collectively, this information was similar to that obtained from microbial and morphologic 110 111 surveys of naturally diseased rats (Giddens, 1968; Nelson 1930a; Klieneberger and Steabben, 1937). These results were inadequate to reach any specific conclusions; however, the disease was reproduced in rats with a known microbiologic background. In evaluating the results of Experiment 1, in which the exposed rats were housed in cages adjacent to diseased rats, pulmonary and tracheal lesions of early CRD as described by Innes gt a1. (1967) were produced in 4 out of 4 rats. In contrast, the rats of Experiments 2 and 3 were housed at a greater distance from the diseased rats and respiratory lesions developed only sporadically. These results were similar to the observations of Bell and Wheeler (1970). They reported a significantly lower incidence of CRD in rats housed at a distance of 4 feet from diseased rats when com- pared to rats housed in the same cage with diseased rats. Aerosol Inoculation Experiments Using a cell-free filtrate (CFF) prepared from the pneumonic lungs of 5 CRD—rats as an inoculum in Experiment 4, and broth cultures of Mycoplasma pulmonis for succeeding eXperiments (Experiments S-ll), respiratory infections were produced on 6 of 7 attempts in germfree rats and on I attempt in ex-germfree rats. Mycoplasma pulmonis was regularly isolated from pneumonic lungs, trachea, and tympanic cavity of the experimentally infected rats. Mor- phologically, the characteristic syndrome of CRD as 112 described by Newberne gt a1. (1961) and Innes at 11. (1967) was observed in the majority of the inoculated rats. Charac- teristic gross lesions were pulmonary consolidation and bronchiectasis. Yellowish muc0purulent exudate filled the lumens of dilated bronchi. Microscopic pulmonary lesions included bronchiectasis, purulent bronchitis, peribronchial infiltration of lymphocytes, and atelectasis. Hyperplastic mucopurulent tracheitis, purulent otitis media, and purulent rhinitis were other characteristic lesions. Adult rats were highly susceptible to the experimental infection and, when compared to the prolonged chronic course of natural cases, the experimental disease was rapid in onset and of short duration. Extensive pneumonia develOped as early as 13 days after exposure. In most cases, consolidation was present in lungs of adult rats killed between 15 and 30 days after inoculation. Only 3 natural deaths were observed, and all occurred during the third week. In contrast to adults, young rats were resistant to the infection and in most cases pulmonary consolidation had not develOped when the rats were killed. Some young rats were maintained for a period of 7 months after inoculation and had not developed lesions whereas adults inoculated at the same time and maintained in the same isolator had extensively consolidated lungs when killed on or before 42 days after inoculation. 113 Observation of the relatively rapid rate of develop— ment of the eXperimentally produced syndrome in adult rats was useful for both research and practical purposes. Experiments on a chronic disease such as CRD might reason- ably be expected to require a year or even longer to complete. The develOpment of a method to rapidly repro— duce the syndrome made it possible to conduct several generations of experiments in an interval that might other- wise have accommodated a single experiment. This capability was especially significant in research utilizing costly germfree techniques. The marked susceptibility and rapid progression of the infection in adult rats may help to explain the sudden outbreaks of respiratory disease that frequently occur after the addition of new rats to an established colony. The episode of acute respiratory disease primarily affects the recently acquired rats making it appear that diseased stock had been obtained. It now appears, however, that the new rats were probably free of respiratory disease and acquired a M; pulmonis infection from chronically diseased rats already present in the colony. The rapidly progressive form of the infection then ensued in these susceptible rats. Usually several deaths occur over an interval of l or 2 weeks following the initial deaths. The epizootic then subsides as the infection assumes the more familiar chronic course of CRD. 114 Klieneberger (1940), Nelson (1967), and Giddens (1968) noticed that young rats seldom develop pulmonary lesions. Giddens mentioned in particular that "research in which weanling rats were inoculated intranasally with one large exposure to organ- isms and then held for 3 or 4 weeks should not be taken too seriously in evaluating the pathogenesis of a particular organism if the results are negative." The natural resistance of the young rat to M;_pu1monis was strikingly apparent in Experiments 4 and 8. Although this resistance of young rats was not an original observation, the present observation under well defined conditions does forcefully reinforce the earlier observations. Experiments of the type described in the quotation from Giddens (1968) and similar short term experiments had been commonplace in CRD research, including Experiments 1—3 reported in this thesis and several conducted by Giddens (1968). Pathology and Etiology The hypothetical separation of CRD of rats into two distinct diseases (Nelson, 1946a) has motivated patholo— gists to search for the corresponding clinicopathologic entities. In particular, attempts have been made to describe the morphologic characteristics of the typical pulmonary syndrome (Innes, gt_gt., 1956; Newberne gt_gl,, 1961) for which a viral causative agent has been postulated (Nelson, 1946a). Remarkably, both Innes gt gt. (1956) and Newberne gt gt. (1961) disregarded or failed to mention any 115 concurrent inflammatory disease in other portions of the respiratory system, although Giddens gt gt. (1971a) in a study of CRD in rats from 3 different origins found that rhinitis and tracheitis were more prevalent than pulmonary lesions in all 3 groups of rats. It is highly unlikely that the rats examined by Innes gt gt. and by Newberne gt gt. were in all cases devoid of tracheal and nasal lesions since several hundred rats were examined by the authors. In fact, Innes gt gt. (1956) stated in a footnote that their "descriptions almost certainly cover the chronic effects of both infectious catarrh and murine pneumonia." Yet with no apparent attempt to discriminate between the pulmonary lesions resulting from infectious catarrh and the lesions due to a virus, Innes gt_gt, described a chronic pulmonary syndrome in rats which they attributed to a viral cause. Newberne gt gt. (1961) also described pulmonary lesions which they ascribed to a viral cause while at the same time acknowledging the presence of infectious catarrh in some of the rats that they studied. Giddens gt_gt, (1971a, 1971b) studied both the upper respiratory and pul- monary lesions of CRD in rats, and attempted unsuccessfully to isolate a virus from CRD-affected rats. They expressed doubt in the validity of a viral etiology for the pulmonary lesions of CRD and proposed an alternate hypothesis in which the pulmonary lesions of CRD were envisioned as "a complex disease initiated by a variety of interrelated factors." Chronic irritation of the bronchial mucosa was 116 essential to this etiologic scheme. It is quite clear that the characteristic lesions of a chronic virally induced pulmonary syndrome in the rat have yet to be described. Dr. John B. Nelson from his research and interest spanning nearly 40 years contributed more to the knowledge of CRD in both rats and mice than any other investigator. However, if the conclusions from research reported here and those reported recently by Kohn and Kirk (1969) and Lindsey gt_gt, (1971) are correct, then Nelson's conclusion that a virus is one of the primary causative agents of CRD can not be supported. Nelson (1967) concluded that growth of mycoplasmas in artificial media results in morphologic and physiologic changes "including loss of virulence." The results of the experiments reported in this thesis strongly refute his conclusion that Mt_pulmonis cannot be cultivated tg_ztttg_without loss of virulence. The above example of a conflict between observations reported in this thesis and a conclusion arrived at by Nelson implies that his techniques and conditions of cultivating mycoplasmas were less than optimum. It was observed in the course of our research, although not tested under controlled conditions, that successful isolation of Mt_pulmonis was enhanced when the specimens from rats were cultured initially in broth medium and subsequently transferred to agar medium for detection and identification of the organisms. Nelson (1967) recommended cultural examination of specimens on agar medium. The agar medium Nelson (1967) used was heart 117 infusion agar (pH 8) with 20% horse serum and penicillin. Shepard and Lunceford (1965) and Hayflick and Stambridge (1967) reported that the optimum pH of the medium is not 7.6 to 8.0 as is often recommended, but 6.8 to 7.4. Hayflick (1965) reported the addition of fresh bakers' yeast extract resulted in enhanced growth of mycoplasmas. The media used in this research met or exceeded the recommendations of Hayflick (1969). The difficulty that can be encountered in the isolation and propagation of the mycoplasmas is well known as was illustrated by a report that apprxoimately 70% of the mycoplasmas isolated from human clinical materials could not be subcultivated after initial isolation (Hayflick and Stanbridge, 1967). Lindsey gt gt. (1971) employed several strans of Mt pulmonis in their research, including a strain originally isolated from mice supplied by Nelson, and Strain M/J-69 supplied by the author. The strain supplied by Nelson in the form of infected lungs and exudates from mice produced rhinitis in 18 of 26 rats but no other lesions of the res- piratory tract or middle ears. The strain supplied by the author was decidedly the most pathogenic strain investigated in their study, producing lesions in all parts of the res- piratory tract and middle ears. The organism was identified by Lindsey gt 1. as Mt_pulmonis by an immunofluorescence technique thus confirming the earlier identification by the author using a growth-inhibition test. 118 The differences in pathogenicity of Mt pulmonis from different sources was amply demonstrated by Lindsey gt gt. (1971); however, many variables other then the source of the isolate may have caused these differences. Notably, differences in tg Xtttg_procedures may have been equally significant. In work with Mt_pneumoniae, Pollack gt gt. (1969) and Low (1971) reported the influence of various constituents of the media on pathogenicity including the effect of pH (Pollack gt gt., 1969). Likewise, various factors may influence the susceptibility of the rat and in this regard Grewal (1971) has reported that semistarved rats were less susceptible to naturally occurring CRD than were fed rats. As mentioned earlier, the virus postulated by Nelson (1948b) has as yet not been isolated. Gay (1967) studied the fine structure of the rat pneumonia agent (Nelson's virus) and the gray-lung agent (gray-lung virus) and con- cluded that both agents resembled mycoplasmas. He was, however, unable to isolate the organisms 12.21EE2.t° confirm his morphologic observations. The respiratory disease produced in germfree and ex- germfree rats in these experiments was essentially the same as the CRD syndrome that has been widely described in rats. Adult rats were highly susceptible to the experimental disease and thus were more suitable experimental subjects than young rats. In reviewing the literature the debatable role of a virus in the CRD syndrome was apparent. The 119 evidence in support of a viral cause was not convincing when evaluated on the basis of current knowledge and tech- nological development. The results of the experiments reported here and those of Kohn and Kirk (1969), Kohn (1971a), and Lindsey gt gt. (1971) indicate that Mt pulmonis is probably the primary causative agent of CRD in rats. If this conclusion is confirmed by future studies, then a more appropriate name for this disease of rats would be respira- tory myc0plasmosis. SUMMARY A total of 142 germfree and ex-germfree rats were used in 11 trials to reproduce experimentally chronic respiratory disease (CRD) of rats, and to isolate and identify the etiologic agent. The first 3 trials were designed to evalu- ate natural transmission of CRD from diseased rats, that had been collected from 5 different sources, to ex-germfree rats. These experiments were followed by 8 trials using germfree and ex-germfree rats to experimentally reproduce CRD, using either a cell-free filtrate prepared from pneumonic lungs or broth cultures of Mycoplasma pulmonis. Following exposure to the filtrate or culture, the rats were killed at selected intervals and examined for microbial and morphologic evidence of CRD. Respiratory lesions typical of the natural disease developed in adult rats within 2 to 6 weeks following inocu— lation. Young rats were resistant to infection and developed lesions only after prolonged incubation, if at all. In one experiment, young rats were maintained for 7 months after inoculation without developing pulmonary lesions. The clinical signs in experimentally infected rats were rough hair coat, dyspnea, anorexia, loss of weight, and humped posture. 120 121 All principal clinical signs and gross and microscopic lesions of CRD were reproduced in 32 of 54 adult germfree and ex-germfree rats. Gross lesions were pneumonia characterized by red and gray areas of consolidation and bronchiectatic bronchi containing mucopurulent or caseopurulent exudate. Microscopic lesions included bronchiectasis, bronchial epi- thelial hyperplasia and necrosis, mucopurulent bronchial exudation, peribronchial and perivascular lymphoid hyper- plasia, and pulmonary atelectasis. Additional lesions were tracheitis, otitis media, and rhinitis. Mycoplasma pulmonis was re-isolated from 40 of 65 lungs, 13 of 13 tympanic cavities, and 8 of 8 tracheas of germfree or ex—germfree rats following inoculation with aerosols of cell-free filtrate or broth cultures of Mycoplasma pulmonis. The organism was identified by a growth inhibition test. It is concluded that Mycoplasma pulmonis is the primary cause of CRD in rats and the apprOpriate name for the disease is respiratory mycoplasmosis. REFERENCES REFERENCES Andrews, C. H.: Viruses gt_Vetebrates. The Williams and Wilkins Co., Baltimore, (1964): 341. Andrews, C. H. and Glover, R. E.: Grey lung virus: an agent pathogenic for mice and other rodents. Brit. J. Exp. Path., 26, (1945): 379—386. Andrews, C. H. and Niven, Janet S. F.: A virus from cotton rats; its relation to grey lung virus. Brit. J. Exp. Path., 31, (1950): 773-778. Baer, H.: Diplococcus pneumoniae type 16 in laboratory rats. Can. J. Comp. Med. Vet. Sci., 31, (1967): 216-218. Beaver, D. L., Ashburn, L. L., and McDaniel, E. G.: Lipid deposits in the lungs of germfree animals. Arch. Path. 76, (1963): 565-570. Bell, D. P., and Elmes, P. C.: Effects of certain organisms associated with chronic respiratory disease on SPF and conventional rats. J. Med. Microbiol., 2, (1969): 511— 519. Bell, D. P., and Wheeler, S. M.: Susceptibility of caesarian- derived rats to natural infection of chronic respiratory disease. Lab. Animals, 4, (1970): 45-53 Brennan, P. C., Fritz, T. E., and Flynn, R. J.: The role of Pasteurella pneumontropica and Mycoplasma pulmonis in murine pneumonia. J. Bacteriol., 97, (1969a): 337-349. Brennan, P. C., Fritz, T. E., and Flynn, R. J.: Murine pneumonia: A review of the etiologic agents. Lab. Anim. Care, 19, (1969b): 360-371. Burek, J. D.: Experimental respiratory infections in the rat. M.S. Thesis, (1970), Michigan State University. Burek, J. 0., Jersey, G. C., and Whitehair, C. K.: Experi- mental respiratory infections in the conventional and germfree rat. Proc. Assoc. for Gnotobiotics, Notre Dame, Ind., June 8-10, 1970. 122 123 Carter, G. R.: Outline of Veterinary Bacteriology and Mycology. Lucus‘Brothers Pub., Gelumbia, Missouri, Cheng K. K.: The eXperimental production of bronchiectasis in rats. J. Path. Bact., 67, (1954), 89-98. Clyde, W. A.: MyCOplasma species identification based upon growth inhibition by specific antisera. J. Immunol., 92, (1964): 958-965. DelGuidice, R. A., Robillard, N. F., and Carski, T. R.: Immunofluorescence identification of mycoplasma on agar by use of incident illumination. J. Bact., 93, (1967): 1205-1209. Dienes, L.: Further observations on the L organisms of Klieneberger. Proc. Soc. Exp. Biol. Med., 39, (1938): 365-367. Edward, D. G., and Freundt, E. A.: The classification and nomenclature of organisms of the pleuropneumonia group. J. Gen. Microbiol., 14, (1956): 197-207. Flynn, R. J., Brennan, P. C., and Fritz, T. E.: Pathogen status of commercially produced laboratory mice. Lab. Anim. Care, 15, (1965): 440-447. Ford. T. M.: An outbreak of pneumonia in laboratory rats associated with Diplococcus pneumoniae type 8. Lab. Anim. Care, Panel, 15, (1965): 448-451. Foster, H. L.: Large scale production of rats free from commonly occurring pathogens and parasites. Proc. Anim. Care Panel, 8, (1958): 92-100. Fuller, R.: The routine microbiological control of germ- free isolators, in The Germ-Free Animal tg Research (ed. by Coates, M. E.). Academic Press, London and New York, 1968. Ganaway, J. R., and Allen, A. M.: Chronic murine pneumonia of laboratory rats: production and description of pulmonary-disease-free rats. Lab. Anim. Care, 19, (1969): 71-79. Gay, F. W.: Fine structure and location of the mycoplasma- like gray lung and rat pneumonia agents in infected mouse lung. J. Bact., 94, (1967): 2048-2061. 124 Gay, F. W., and Attridge, J. T.: The fine structure of cytOplasmic inclusions in a myc0plasma-like infection of mice. J. Cell Sci., 2, (1967): 445-450. Giddens, W. E., Jr.: The pathology of chronic respiratory disease in the rat. Ph.D. Thesis (1968), Michigan State University. Giddens, W. E., Jr., Keahey, K. K., Carter, G. R., and Whitehair, C. K.: Pneumonia in rats due to infection with Corynebacterium kutscheri. Path. Vet., 5, (1968): 227-237. Giddens, W. E., Jr., Whitehair, C. K., and Carter, G. R.: Morphologic and microbiologic features of nasal cavity and middle ear in germfree, defined-flora, conventional, and chronic respiratory disease-affected rats. Am. J. Vet. Res., 32, (1971a): 99-114. Giddens, W. E., Jr., Whitehair, C. K., and Carter, G. R.: Morphologic and microbiologic features of trachea and lungs in germfree, defined-flora, conventional, and chronic respiratory disease-affected rats. Am. J. Vet. Res., 32, (1971b): 115-129. Gordon, H. A., and Pesti, L.: The gnotobiotic animal as a tool in the study of host microbial relationship. Bacteriol. Rev., 35, (1971): 390-429. Grewal, T.: Adaptive responses to semistarvation and re- feeding in rats. Ph.D. Thesis (1971), Michigan State University. Habermann, R. T., Williams, F. P., Jr., McPherson, C. W., and Every, R. R.: The effect of orally administered sulfamerazine and Chlortetracycline on chronic respiratory disease in rats. Lab. Anim. Care, 13, (1963): 28-40. Hayflick, L.: Tissue cultures and mycoplasmas. Texas Rep. Biol. Med., 23, (1965): 285-303. Hayflick L.: The Mycgplasmatales and the L-Phase gt Bacteria. Meredith Corporation, New York, 1969 Hayflick, L., and Stanbridge, E., Isolation and identifi- cation of mycoplasma from human clinical material. Ann. N.Y. Acad. Sci., 143, (1967): 608-621. Hektoen, L.: Observations on pulmonary infection in rats. Trans. of the Chicago Path. Soc., (1916): 105-108. 125 Heyl, J. G.: A study of pasteurella strains from animal sources. Antonie V. Leeuwenhoek. Microbiol., 29, (1963): 79-83. Hoag, W. G., Wetmore, P. W., Rogers, J., and Meier, H.: A study of latent pasteurella infection in a mouse colony. J. Inf. Dis., 111, (1962): 135-140. Hoskins, H. P., and Stout, A. L.: Bacillus bronchisepticus as the cause of an infectious respiratory disease of the white rat. J. Lab. Clin. Med., 5, (1919-20): 307-320. Innes, J. R. M., Garner, F. M., and Stookey, J. L.: Respira- tory disease in rats, in Pathology of Laboratoty Rats ggg_Mice (ed. by Cotchin, E., and R55, F. J. C.). F. A. Davis Co., Philadelphia, 1967. Innes, J. R. M., McAdams, A. J., and Yevich, P.: Pulmonary disease in rats. A survey with comments on "chronic murine pneumonia". Am. J. Path., 32, (1956): 141-160. Jawetz, E.: A pneumotropic Pasteurella of laboratory animals. I. Bacteriological and serological characteristics of the organism. J. Inf. Dis., 86, (1950): 172-183. Jawetz, E., and Baker, W. H.: A pneumotropic Pasteurella of laboratory animals. II. Pathological and immunological studies with the organism. J. Inf. Dis., 86, (1950): 184-196. Jericho, K. W. F.: Pathogenesis of pneumonia in pigs. Vet. Rec., 82, (1968): 507-517. Jones, F. S.: The source of the microorganisms in the lungs of normal animals. J. Exper. Med., 36, (1922): 317-328. Joshi, N. N., Dale, D. G., and Blackwood, A. C.: Etiology of chronic endemic pneumonia. Rev. Canad. Biol., 24, (1965): 169-178. Kellogg, T. F. and Wostmann, B. 8.: Stock diet for colony production of germfree rats and mice. Lab. Anim. Care, 19, (1969): 812-814. Klein, E.: Discussion of the Pathological Society of London, Lancet, 1, (1903): 238-239. Klieneberger, E.: The natural occurrence of PPLO in apparent symbiosis with Streptobacillus moniliformis and other bacteria. J Path. Bact., 40, (1935): 93-105. Klieneberger, E.: The pleurOpneumonia-like organisms: Further comparative studies and a descriptive account of recently discovered types. J. Hyg., (London), 40, (1940): 204-222. 126 Klieneberger-Nobel, E.: Pleuropneumonia-like Organisms (PPLO): Mycoplasmataceae. Academic Press, London/ New York, 1961. Klieneberger-Nobel, E., and Cheng, K. K.: On the associa- tion of the pleuropneumonia-like L3 organism with ex- perimentally produced bronchiectasis in rats. J. Path. Bact., 70, (1955): 245-246. Klieneberger, E., and Steabben, D. B.: On a pleurOpneumonia- like organism in lung lesions of rats with notes on the clinical and pathological features of the underlying conditions. J. Hyg., (London), 37, (1937): 143-152. Klieneberger, E., and Steabben, D. E.: On the association of the pleuropneumonia-like organism L3 with bronchi- ectatic lesions in rats. J. Hyg., (London), 40, (1940): 223-227. Kundsin, R. B.: Aerosols of mycoplasmas, L forms, and bacteria: Comparison of particle size, viability, and lethality of ultraviolet radiation. Appl. Microbiol., 16, (1968): 143-146. Kutscher, Dr.: Ein Beitrag zur Kenntniss der bacillaren Pseudotuberculose der Nagethiere. Z. Hyg. Infktkr., 18, (1894): 327-342. Kohn, D. F.: Sequential pathogenicity of Mycoplasma pulmonis in laboratory rats. Lab. An. Sci., 21, (1971a): 849-855. Kohn, D. F.: Bronchiectasis in rats infected with Mycoplasma pulmonis: an electron microscopy study. Lab. An. Sci., (1971b): 856-861. Kohn, D. F., and Kirk, B. E.: Pathogenicity of Mycgplasma pulmonis in laboratory rats. Lab. Anim. Care, 19, (1969): 321-330. Laurenzi, G. A., and Guarneri, J. J.: Effects of bacteria and viruses on ciliated epithelium. A study of the mechanisms of pulmonary resistance to infection: The relationship of bacterial clearance to ciliary and alveolar macrophage function. Amer. Rev. Resp. Dis. Suppl, 93, (1966): 134-141. LaMaistre, C., and Thompsett, R.: The emergence of pseudo- tuberculosis in rats given cortisone. J. Exper. Med., 95, (1952): 393-408. Lindsey, J. R., Baker, H. J., Overcash, R. G., Cassell, G. H., and Hunt, C. E.: Murine chronic respiratory disease, Amer. J. Path., 64, (1971): 675-708. 127 Low, I. E.: Effect of medium on H202 levels and peroxidase- like activity by Mygoplasma pneumoniae. Infect. Immun., 3, (1971): 80-86. Luna, L. G. (editor): Manual gt Histologic Staining Methods gt the Armed Forces Institute gt_Pathology. Third’ edition. New York, McGraw-Hill Book Company, 1968. Lutsky, I. I., and Organick, A. B.: Pneumonia due to myco- plasma in gnotobiotic mice. I. Pathogenicity of Mycoplasma pneumdniae, Mycoplasma salvarium, and Mycgplasma pulmonis for—thefilungs of conventional and gnotobiotic mice. J. Bact., 92, (1966): 1154-1163. Lutsky, I. I., and Organick, A. B.: Recovery of mycoplasma from gnotobiotic systems. Lab. Anim. Care, 18, (1968): 610-615. Macalister, G. H., and St. John Brooks, R.: cited by Klieneberger, E., and Steabben, D. B., 1937. Marmion, B. P., and Hers, J. F. P.: The mycoplasma (PPLO) agents: Observations on Eaton primary atypical pneumonia agent and analogous problems in animals. Am. Rev. Resp. Dis., 88, (1963): 198-211. McCordock, H. A., and Congdon, C. C.: Suppurative otitis of the albino rat. Proc. Soc. Exper. Biol. Med. N. Y., 22, (1924): 150-154. Mirick, G. S., Richter, C. R., Schaub, I. B., Franklin, R., MacCleary, R., Schipper, G., and Sptiznaged, J.: An epizootic due to pneumococcus type II in laboratory rats. Am. J. Hyg., 52, (1950): 48-53. Nelson, J. B.: The reaction of the albino rat to the intra- aural administration of certain bacteria associated with middle ear disease. J. Exper. Med., 52, (1930): 873- 883. Nelson, J. B.: Infectious catarrh of mice. I. A natural outbreak of the disease. J. Exper. Med., 65, (1937a): 833-842. Nelson, J. 8.: Infectious catarrh of mice. II. The detec- tion and isolation of coccobacilliform bodies. J. Exper. Med., 65, (1937b): 843-850. Nelson, J. B.: Infectious catarrh of mice. III. The etiological significance of the coccobacilliform bodies. J. Exper. Med., 65, (1937c): 851-860. 128 Nelson, J. B.: Infectious catarrh of the albino rat. I. Experimental transmission in relation to the role of Actinobacillus muris. J. Exper. Med., 72, (1940a): 645-654. Nelson, J. B.: Infectious catarrh of the albino rat. II. The causal relation of coccobacilliform bodies. J. EXper. Med., 72, (1940b): 655-662. Nelson, J. B.: Reciprocal transmission tests with infec- tious catarrh of chickens, mice, and rats. J. Exper. Med., 76, (1942): 253-262. Nelson, J. B.: Studies on endemic pneumonia of the albino rat. I. The transmission of a communicable disease to mice from naturally infected rats. J. Exper. Med., 84, (1946a): 7-14. Nelson, J. B.: Studies on endemic pneumonia of the albino rat. II. The nature of the causal agent in experimentally infected mice. J. Exper. Med., 84, (1946b): 15-23. Nelson, J. B.: Studies on endemic pneumonia of the albino rat. III. Carriage of the virus-like agent by young rats and in relation to susceptibility. J. Exper. Med., 87, (1948a): 11-19. Nelson, J. B.: The nasal transmission of pleuropneumonia- like organisms in mice and rats. J. Inf. Dis., 82, (1948b): 169-176. Nelson, J. B.: Observations on a pneumotropic virus obtained from wild rats. I. Transmission of the agent to white mice and rats. J. Inf. Dis., 84, (1949a): 21-25. Nelson, J. B.: Observations on a pneumotropic virus obtained from wild rats. II. Biological characteristics of the agent. J. Inf. Dis., 84 (1949b): 26-31. Nelson, J. B.: Studies on endemic pneumonia of the albino rat. IV. Development of a rat colony free from respiratory infections. J. Exper. Med., 94, (1951): 377-386. Nelson, J. B.: Chronic respiratory disease in mice and rats. Proc. Anim. Care Panel, 6, (1955): 9-15. Nelson, J. B.: The etiology and control of chronic respira- tory disease in the rat. Proc. Anim. Care Panel, 7, (1957): 30-40. Nelson, J. B.: Chronic respiratory disease in mice and rats. Lab. Anim. Care, 13, (1963): 137-143. 129 Nelson, J. B.: Respiratory infections of rats and mice with emphasis on indigenous mycoplasmas, in Pathology of Laboratornyats and Mice (ed. by Cotchin, E., and—— Roe, F. J. C.). F. A. Davis Co., Philadelphia, 1967. Nelson, J. B., and Gowen, J. W.: The incidence of middle ear infection and pneumonia in albino rats at different ages. J. Inf. Dis., 46, (1930): 53-63- Nelson, J. B., and Gowen, J. W.: the establishment of an albino rat colony free from middle ear disease. J. Exper. Med., 54, (1931): 629-636. Newberne, P. M., Salmon, W. D., and Hare, W. V.: Chronic murine pneumonia in an experimental laboratory. Arch. Nocard, E., and Roux, E. R.: Avec la collaboration de M. M. Borrel, Salimbeni et Dujardin-Beaumetz. Le microbe de la peripneumonie. Ann. Inst. Pasteur, 12, (1898): 240. Organick, A. B., and Lutsky, I. I.: Pneumonia due to mycoplasma in gnotobiotic mice. IV. Localization and identification of Mygoplasma pulmonis in the bronchi of infected gnotobiotic mice by immunofluorescence and by light microscopy. J. Bact., (1968): 250-258. Organick, A. B., Siegesmund, K. A., and Lutsky, I. I.: Pneumonia due to mycoplasma in gnotobiotic mice. II. Localization of Mycoplasma pulmonis in the lungs of infected gnotobiotic mice by electron microscopy. J. Bact., 92, (1966): 1164-1176. Pankevicus, J. A., Wilson, C. E., and Farber, J. F.: The debatable role of a pleuropneumonia-like organism in the etiology of chronic pneumonia in rats. Cornell Vet., 47, (1957): 317-325. Passey, R. D., Leese, A., and Knox, J. G.: Bronchiectasis and metaplasia in the lung of the laboratory rat. J. Path. Bact., 42, (1936): 425-434. Petrie, G. F., and Macalister, G. H.: Report of the Local Government Board, London, 1911, Appendix A, page 91. Pollack, J. D., Somerson, N. L., and Senterfit, L. B.: Effect of pH on the immunogenicity of MyCOplasma pulmoniae. J. Bact., 97, (1969): 612-619. Sacquet, E.: General technique for maintaining germ-free animals, in The Germ-Free Animal tg Research (ed. by Coates, M. E.)T' Academic Press, London and New York, 1968. 130 Shepard, M. C., and Lunceford, C. D.: Effect of pH on human mycoplasma strains. J. Bact., (1965): 265-270. Smith, D. T., Bethune, N., and Wilson, J. L.: Etiology of spontaneous pulmonary disease in the albino rat. J. Bact., 20, (1930): 361-370. Strangeways, W. I.: Rats as carriers of Streptobacillus moniliformis. J. Path. Bact., 37, (1933): 45-51. Trexler, P. G.: The use of plastics in the design of isolator systems. Ann. N. Y. Acad. Sci., 78, (1959): 29-36. Tunnicliff, R. J.: Streptothrix in bronchopneumonia of rats similar to that of rat-bite fever. J. Inf. Dis., 19, (1916): 767-772. Ventura, J., and Domaradzki, M.: Pathogenesis of eXperi- mental bronchiectasis in laboratory rats. Arch. Path. 83, (1967a): 80-85. Ventura, J., and Domaradzki,M.: Role of mycoplasma infec- tion in the development of experimental bronchiectasis in the rat. J. Path. Bact., 93, (1967b): 342-348. Vrolijk, H., Verlinde, J. D., and Braoams, W. G.: Virus pneumonia (snuffling disease) in laboratory rats and wild rats due to an agent probably related to grey lung virus of mice. Ant. V. Leeu., 23, (1957): 173-183. Weisbroth, S. H., and Freimer, E. H.: Laboratory rats from commercial breeders as carriers of pathogenic pneumococci. Lab. Anim. Care, 19, (1969): 473-478. Wheater, D. W. F.: The bacterial flora of an SPF colony of mice, rats, and guinea-pigs, in Husbandry of Laboratory Animals (ed. by Conalty, M. L.). Academic—Fress, London and New York, 1967. Winsser, J.: A study of Bordetella bronchiseptica. Proc. Anim. Care Panel, 10, (1960): 87-101. Zucker, T. F., Zucker. L. M., and Seronde, J.: Antibody formation and natural resistance in nutritional deficiencies. J. Nutr., 59, (1956): 299-308. VITA The author was born at Highland Park, Michigan, on August 20, 1940. He received his primary and secondary education in the public schools of Romeo, Michigan. After graduation from high school in 1958, he continued his education at Eastern Michigan University, Ypsilanti, Michigan, and received a Bachelor of Arts degree in February, 1964. He entered Michigan State University in September, 1963, and was awarded a Bachelor of Science degree in June, 1965, and a Doctor of Veterinary Medicine degree in June, 1967. With the support of the Upjohn Fellowship he entered the Department of Pathology at Michigan State University as a Master's degree candidate in June, 1967, and was awarded the Master of Science degree in June, 1969. From December, 1968, to July, 1972, the author held temporary appointments as an Instructor in the Department of Pathology, Michigan State University. During this time, he worked toward completion of the Doctor of Philosophy degree in Pathology. The author married Miss Barbara Jean Teller in 1958. They have two daughters: Cynthia Rhoda and Kay Elizabeth. 131 "Tlfl'l‘t‘lfllluslflflfifitflflfllflfljljfliflifllmllflmms 183