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Mvmn: . . . ‘ $a'h: "“7 £;‘h:~£:e;‘*:¢: OOD'JJ: " M?::1.' ‘0' *v' 7:1“4- #3 N IA}. 3 ‘ ' - o . a 0‘ "l B ‘ by," {Lye-.1... w“'.!'0"‘l“_ Duo n"m':. .QQ-‘pfivmo- DIP...“ H-{Ei “an . n W M ”y fr”?! - a an” '49 'm “ “yo-v.0 ""(‘i‘ "r"?- ~HV-n-w“ lo- 'rrvu'n” w !;'o':é.'s .':S~ «an: 23‘1”:er o“. ’nLWA-h-m-t- ABSTRACT MYCOPLASMA PULMONIS INFECTION OF THE RESPIRATORY TRACT AND MIDDLE EAR IN THE RAT By Robert B. Gibson Research was conducted to reproduce.Mycop1asma pulmonis infec— tion in rats reared conventionally, exgermfree, and germfree. Ninety- six rats were used in S sequential experiments. Chronic respiratory disease was reproduced in rats exposed to broth cultures of M. pulmonis. The clinical signs and lesions were characteristic of the natural disease. Clinical signs were observed in the rats by 3 weeks after exposure to M. pulmonis. The most severe clinical signs were seen in the germfree rats. These rats developed dyspnea and became depressed. The young conventionally reared rats, exposed to M. pulmonis at 45 days of age, developed only mild clinical signs. The mild signs were often followed by apparent recovery. Tracheitis, rhinitis, and otitis media were present in rats exposed to M. pulmonis regardless of age and method of rearing. The tracheitis and rhinitis were most severe in rats exposed at 45 days of age to both M. pulmonis and ammonia fumes. Also, upper respiratory lesions and purulent exudate in the middle ear were observed in the conventional rats exposed to only ammonia fumes. Robert B. Gibson Bronchopneumonia with pulmonary solidification (consolidation) was a consistent finding in conventionally reared, exgermfree, and germfree rats exposed to M. pulmonis at 3 months of age or older. Conventional raised rats exposed to M. pulmonis alone and to M. pulmonis and ammonia fumes at 45 days of age developed fewer lung lesions. Upper respiratory and middle ear changes but no significant lung lesions were observed following exposure of 45-day-old conven— tional rats to ammonia fumes only. Mycoplasma pulmonis microorganisms were reisolated from pre— viously exposed rats. The reisolation procedures were complicated by the presence of contaminating microorganisms in the conventional and exgermfree rats. Chronic respiratory disease in the rats was primarily due to M. pulmonis. Ammonia fumes and contaminating microorganisms may con- tribute to the disease as it occurs naturally in rat colonies used in research. MYCOPLASMA PULMONIS INFECTION OF THE RESPIRATORY TRACT AND MIDDLE EAR IN THE RAT BY c L“. Robert Bi Gibson A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pathology 1976 ACKNOWLEDGEMENTS- The author wishes to express his appreciation to the following: To Dr. C. K. Whitehair, my major professor, for counseling and guidance in the conduct of this research. To Dr. R. F. Langham for counsel and excellent training in pathology. To Dr. G. R. Carter for providing facilities and guidance on microbiologic methods and techniques. To the Department of Pathology and College of Veterinary Medicine for providing facilities, technical assistance, and the financial support to pursue graduate study. To especially my wife, Carol, for encouragement, support and understanding during my tenure of graduate training. ii TABLE OF CONTENTS INTRODUCTION . C O O O O O C O O O O O O O C O O O . . . LITERATUM RWIEW O O O O O O O O I O O O O O C O O O O Mycoplasmal Diseases of Man and Animals. . . . . Mycoplasma pulmonis Infection of Rats. . . . . . Factors Contributing to the Susceptibility of Rat to Respiratory Mycoplasmosis . . . . . . . . . Clinical Signs and Lesions . . . . . . . . . . . Methods of Control and Prevention. . . . . . . . Immune Response of Rats to.Myccplasma pulmonis . Germfree Principles. . . . . . . . . . . . . . . Summary of Literature Review . . . . . . . . . . OBJECTIVES O O O Q C O O O O O O O O O O O O O O O O O 0 MATERIALS AND MHODS I C O C O O O O O O Q 0 O O O O 0 RESULTS Sources and Care of Experimental Animals . . . . Necr0psy and Tissue Preparation Procedures . . . Experimental Exposure of the Rats. . . . . . . . Microbiologic Techniques . . . . . . . . . . . . Experiments. . . . . . . . . . . . . . . . . . . Experiment 1. . . . . . . . . . . . . . . Experiment 2. . . . . . . . . . . . . . . Experiment 3. . . . . . . . . . . . . . . Experiment 4. . . . . . . . . . . . . . . Experiment 5. . . . . . . . . . . . . . . Colony Controls . . . . . . . . . . . . . Summary of Experiments . . . . . . . . . . . . . Experiment 1. . . . . . . . . . . . . . . Experiment 2. . . . . . . . . . . . . . . Experiment 3. . . . . . . . . . . . . . . Experiment 4. . . . . . . . . . . . . . . Experiment 5. . . . . . . . . . . . . . . Colony Controls . . . . . . . . . . . . . iii Page 11 13 14 16 17 18 18 19 20 21 22 22 23 23 23 24 24 25 25 25 28 29 29 31 31 Composite Description of CRD DISCUSSION. . SUMMARY.. . . REFERENCES. . VITA. Clinical Signs. . . Gross Lesions . . . Microscopic Lesions Microbiology. . . . iv Page 32 32 35 35 48 49 52 54 58 LIST OF TABLES Table Page 1 Experimental design, lesions and Mycoplasma pul- monis isolations . . . . . . . . . . . . . . . . . . . . 26 Figure 10 11 12 LIST or FIGURES ' Clinical signs of CRD in a 55-day-old rat reared germfree, 25 days after exposure to a broth culture of M. pulmonis . . . . . . Dorsal view of lungs in a 9-month-old, conven- tionally reared rat, 6 months after aerosol ex- posure to M. pulmonis. . . Bronchiole and surrounding tissue of a 3-monthrold, exgermfree rat . . . . . . Subacute bronchiolitis showing peribronchiolar lymphocytic infiltration in a 4-month-old, conven- tionally reared rat, 29 days after exposure to M. pulmonis. . . . . . . . Bronchiolitis showing a respiratory bronchiole, dilated and filled with exudate, 40 days after exposure of an exgermfree rat to M. pulmonis . . Tracheitis in a 95-day-old, conventionally reared rat exposed to M. pulmonis and ammonia fumes at 45 days of age. . . . . . . . Higher magnification of Figure 6 . . . Purulent rhinitis in an exgermfree rat that.was exposed to M; pulmonis at 3 months of age and died 42 days later. . . . . . . Purulent rhinitis in a lOZ-day-old, conventionally reared rat that was exposed to M. pulmonis and ammonia fumes at 45 days of age. Higher magnification of Figure 9 . Otitis media in a 4-month-old rat that was reared germfree and exposed to M. pulmonis at 3 months of Higher magnification of the tympanic cavity of the same ear as in Figure 11 . vi age Page 34 34 37 37 40 40 42 42 44 44 46 46 Figure Page 13 Higher magnification of tympanic cavity of same ear as Figures 11 and 12. . . . . . . . . . . . . . . . . . . 4? vii INTRODUCTION Chronic respiratory disease in rats is an important problem and seriously restricts their usefulness in a wide variety of researdh. The disease is difficult to detect clinically in a rat colony. Experi- mental results obtained using rats afflicted with the disease would be unreliable (Lindsey et al., 1971). Viruses, bacterial agents, and combinations of pathogens have been suggested as causes of chronic respiratory disease (CRD) in rats (Brennan et al., 1971). In recent years, however, Mycoplasma pulmonis has been demonstrated to be the primary cause of CRD (Kohn and Kirk, 1969; Lindsey et al., 1971; Whittlestone et al., 1972; Jersey et al., 1973). Research workers have recently suggested that factors altering pulmonary clearance may also play a part in the pathogenesis of CRD in the rat (Cassell et al., 1973). The significance of this disease in rat colonies used for research, the importance of the mycoplasmal microorganisms in diseases of man and livestock, and the possibilities of the rat as a model for respiratory disease research emphasize the need for additional inves- tigations on the role of M; pulmonis pneumonia in rats. Respiratory disease in rats has been referred to by a variety of terms and, for consistency, in this thesis the infection will be called chronic respiratory disease. LITERATURE REVIEW The literature on CRD in the rat has been recently reviewed rather extensive1y by other workers (Nelson, 1967; Brennan et al., 1969; Giddens et al., 1971a,b; Cassell et al., 1973). Therefore, this review is focused primarily on the cause, factors that contribute to susceptibility, the immune response, clinical signs and lesions, pathogenesis of infection, and methods of prevention and control of M. pulmonis in rats. Germfree techniques that enhance researdh on the pathogenesis and in control and prevention of the disease are also included. Mycoplasmal Diseases of Man and Animals The determination that.Mycop1asma.mycoideS was the cause of contagious bovine pleuropneumonia by Nocard and Roux in 1898 brought about a systematic study of mycoplasmal diseases in man and animals. During the next six decades only a few diseases caused by mycoplasmas were identified. Increased interest was stimulated in the early 1960's when Mycoplasma pneumoniae was found to be the cause of pri- mary atypical pneumonia in man. Since that time, important diseases caused by mycoplasmas have been identified in numerous domestic Species. Presently mycoplasmas are associated with important diseases in cattle, swine, sheep, goats, fowl, and laboratory animals. Myco- plasmas have been isolated from horses, dogs, and cats, but proof of 2 3 pathogenicity is lacking (Carter, 1975; Whittlestone, 1975). Since it is likely more mycoplasmas will be discovered, further association of these microbes with animal disease will probably occur. Mycoplasmas are the smallest known organisms able to grow and multiply autonomously. These organisms have no cell wall but are bounded by a unit membrane. They can pass through pores and pene- trate into spaces much smaller than their usual diameter. The myco- plasmas have rather fastidious growth requirements (Whittlestone, 1975). The order Mycoplasmatales can be separated into three genera. The first genus, the true mycoplasmas, requires cholesterol as a growth substance. The second genus, the acholeplasmas, does not require cholesterol. The third genus requires urea as an essential metabolite, and members of this genus are called T-mycoplasmas or ureaplasmas. The T-mycoplasmas produce very small colonies. Most of the known pathogenic mycoplasmas belong to the genus of true mycoplasmas (Whittlestone, 1975). Several of the mycoplasmas inhabit the upper respiratory tract, genital tract, and upper digestive tract of animals without causing injury to the host. Pathogenic organisms may involve the respira- tory tract, joints, mammary glands and other body systems as extra— cellular parasites. Infections caused by mycoplasmas are often associated with stresses and are usually low grade and Chronic (Carter, 1975). 4 Mycoplasma pulmonis Infection of Rats While Mycoplasma pulmonis is now considered to be the primary etiologic agent of rhinitis and otitis media commonly associated with the upper respiratory tract of the rat, the role of this organism in the development of lower respiratory infection has been investigated, but a role has not been fully established (Cassell et al., 1973). Klieneberger and Steabben (1937) at the Lister Institute, London, first reported the isolation of a pleuropneumonia—like organism (PPLO) from the lungs of rats with pneumonia. This was named the L1 organism. In a subsequent report (Klieneberger and Steabben, 1940), the L organism (changed from the previously named L 3 organism) was 1 closely correlated with bronchiectatic lesions in laboratory rats, although this organism did not produce bronchiectasis following experimental inoculation. The L3 organism later became known as M. pulmonis. Klieneberger-Nobel and Chang (1955) and later Venture and Domaradzki (1967), physicians at St. Joseph of Rosemont Hospital, Montreal, were able to experimentally produce pneumonia and bronchi- ectasis by ligating bronchi of young rats that were naturally exposed to M. pulmonis. Nelson (1940, 1967), at the Rockefeller Institute, New York, described a relationship between "coccdbacilliform bodies" from grams stained exudates and disease of the upper respiratory tract, middle ear, and infrequently the lungs. The "coccobacillifbrm bodies" were subsequently identified as M. pulmonis. Nelson (1967), in a review summarizing three decades of research on chronic respiratory disease in rats, concluded that the condition consisted of two entities. 5 These were infectious catarrh caused by M. pulmonis and enzootic bronchiectasis caused by a virus. The infectious catarrh resulted in rhinitis, otitis media and less often pneumonia, and the enzootic bronchiectasis was responsible for rhinitis and chronic pneumonia. Brennan et a1. (1969), at Argonne National Laboratory, concluded that the usual causes of murine pneumonia (any pneumonia in rats) were M. pulmonis and Pasteurella multocida. They also believed that the two causative organisms together produced an explosive pneumonia and death. Using M. pulmonis cultures, Kohn and Kirk (1969), at the West Virginia University Medical Center, experimentally produced lung lesions like those of CRD in conventionally reared progeny of gnoto- biotic Sprague-Dawley rats, monocontaminated with lactobacilli. Repeated inoculations were required. Lindsey et a1. (1971) success- fully produced the lesions of natural CRD with various inocula containing only M. pulmonis in inbred Fischer rats raised under rigid disease-free conditions. 0f the five strains of organisms used, the most severe lesions were produced by strain M/J69, isolated earlier and supplied to Dr. Lindsey's group by Dr. George Jersey at Michigan State University. Whittlestone et a1. (1972), a pioneering group of researchers on mycoplasmal diseases at Cambridge University, reported that M. pulmonis by itself could produce pneumonia and bronchiectasis. They used Sprague-Dawley, cesarean derived, barrier sustained rats. A cloned inoculum was used to eliminate possible carryover of another pathogen. The course of the disease was chronic with lung lesions 6 first being observed at 85 days after exposure and persisting to 715 days. Jersey et al. (1973), at Michigan State University, using germ- free rats, concluded that M. pulmonis was the primary cause of Chronic respiratory disease in rats and that an appropriate name for the disease was respiratory mycoplasmosis. The disease deve10ped fairly rapidly with typical respiratory lesions occurring within 2 to 6 weeks following exposure of adult germfree rats. Cassell et a1. (1973) emphasized that Koch's postulates for producing all respiratory tract lesions characteristic of CRD have been satisfied using pure cultures of M; pulmonis. They suggested murine mycoplasma respiratory disease as an appropriate name for the disease. Factors Contributing to the Susceptibility of Rats to Respiratory Mycoplasmosis Reports have indicated that dietary factors, environmental influences, and genetic factors affect the susceptibility of rats to chronic reSpiratory disease. Lindsey et a1. (1971) mentioned the importance of proper nutrition. Tvedten et al. (1973) reported germfree rats fed deficient vitamin A and vitamin E rations were more suscep- tible to M. pulmonis infection than were those fed adequate levels of both vitamins and that high levels of vitamin supplementation may be a means of reducing the disease incidence in rat colonies. Kasali (1974) reported that conventionally reared rats fed a high lard diet were more susceptible to M. pulmonis than those fed a standard commercial diet. 7 Giddens et al. (1971b) noted that environmental factors such as air flow, temperature and humidity of animal rooms, overcrowding, sanitary standards, and concentration of ammonia fumes influence the establishment of tracheobronchial infections in rats. The rapid respiratory rate of the rat, the anatomical structure of the rat bronchus, and genetic susceptibility were mentioned as additional influencing factors. There is evidence that age affects the susceptibility of rats to respiratory mycoplasmosis. Nelson (1967), Giddens et al. (1971b), and Jersey et a1. (1973) all noticed that young rats were more resistant to CRD and seldom developed significant lung lesions. Adult rats, on the other hand, were highly susceptible to experi- mental CRD and rapidly developed an extensive pneumonia. Cassell et a1. (1973) explained that the pulmonary clearance of organisms from the lower respiratory tract is important in the prevention of pulmonary lesions in rats with chronic respiratory disease. They administered hexamethylphosphoramide (HMPA), a com- pound which interferes with normal pulmonary clearance, and markedly enhanced lower respiratory lesions due to M; pulmonis. High levels of sulfur dioxide and ammonia were suggested as playing a part in lowering resistance of rats to Mycoplasma, since both compounds interfere with pulmonary clearance in the rat. Clinical Signs and Lesions Chronic respiratory disease in laboratory rats has been described in the literature since the early 20th century. Klein (1903) gave a brief description of CRD. He described extensive pulmonary 8 consolidation with a fibrinous exudate in the bronchi and alveoli of affected lungs. A diphtheroid bacterium was isolated from the diseased lungs. Hektoen (1916), at the Memorial Institute of Infectious Diseases, Chicago, provided one of the first detailed morphological descrip— tions of CRD. Bronchitis and bronchopneumonia were observed in laboratory rats examined for lung lesions. Two types of pulmonary consolidation were identified. Suppuration was Observed in the first type and a mucoid type of degeneration was seen in the second. Unthriftiness, listlessness, and dirty hair coats were the main clinical Signs noted. A streptothrix was isolated from the lungs. McCordock and Congdon (1924), at the Buffalo General Hospital in New York, outlined the clinical and historical manifestations of suppurative otitis media in laboratory rats. The affected animals tilted their heads to one side and circled in the direction of the tilt. In these rats the squamous epithelial lining of the middle ear was replaced by fibrous granulation tissue, and pus filled the cavity. Osteitis was present in bone surrounding the cavity. Klieneberger and Steabben (1937) reported considerable variation in the gross appearanace of lung lesions in CRD-affected rats. They emphasized that microsc0pic lesions may exist.without the develop— ment of detectable gross lesions. The initial microscopic lung lesions were visualized as a complicated group of simultaneous changes involving the bronchi. The significant changes were pro- liferation of bronchial epithelium, increased mucus production, peri- bronchial lymphocytic infiltration, bronchial dilatation, alveolar collapse, neutroPhilic infiltration through the brondhial epithelium 9 into the lumen, and accumulation of mononuclear cells in the sub- mucosa and lamina propria. As the disease progressed, a bronchial exudate containing neutrophils blocked the lumen. Finally the bronchial epithelium and neutrophils were destroyed leaving a purulent mass surrounded by fibrous connective tissue. Innes et a1. (1956) and Newberne et a1. (1961) have provided a detailed and rather complete description of the gross and microscopic lesions of naturally occurring CRD in rat research colonies. Innes and associates observed that most of the affected rats lacked visible clinical signs although in the advanced disease state weight loss, rough hair coat, and dyspnea were noticeable. Entire lung lobes in affected rats were indurated, rubbery in consistency, and cobbled at the surface. Despite the often diffuse lung involvement, only a low incidence of emphysema and pleural adhesions was observed. An accumulation of inflammatory exudate in the alveolar walls near affected bronchi was noted. This process often accompanied the con- current bronchial changes. Peribronchial collapse was an accompanying finding. Foamy macrophages were often seen in the affected alveoli. Newberne and co-workers emphasized a squamous metaplasia of the affected lung bronchi. Additional pulmonary lesions noted were induration, bronchiectasis, lymphoid proliferation, pneumonia and hyperplasia of bronchial epithelium. Nelson (1967) observed a compen- satory hypertrophy of unconsolidated portions of affected lungs. Giddens et a1. (l97la,b) described the morphologic characteristics of respiratory systems of germfree, conventional and CRD affected rats. The morphology of the lungs, middle ear, trachea and nasal 10 cavity of rats under the three conditions was described. In the natural infection the rhinitis was purulent in nature with exudation in the nasal cavity, a marked proliferation of lymphocytes beneath the epithelium and changes in the mucosal surface which sometimes included necrosis. Lindsey et al. (1971) described in considerable detail the sequential appearance of disease signs in a CRD affected rat colony. Signs of the disease were not observed until the rats were at least 1 month of age. At this time, small encrustations around the external nares were seen and snuffling was noticed. Clinically inapparent otitis media had developed and varying numbers of rats had acquired pneumonia. Most of the lesions up to this time required histologic examination for detection. By 2 months of age, there was polypnea, inactivity, humped posture, rough hair coat, and diminished weight gains. However, in many rats the clinical signs were difficult to detect. Mortality normally remained low. Examples were cited in which the incidence of inapparent infection in adult rats ranged from 50 to 100%. Jersey et a1. (1973), in previous research in this department, were able to readily reproduce CRD by using germfree rats. The clinical signs were essentially the same as reported by previous researchers. Rats exposed under 21 days of age did not develop a rapidly progressive respiratory infection. The lung lesions observed included bronchiectasis, mucopurulent bronchial exudation, peribronchial and perivascular lymphoid hyperplasia, and pulmonary atelectasis. The lung lesions were accompanied by a tracheitis with hyperplasia of the epithelium and an infiltration of lymphocytes, 11 plasma cells, and neutrophils beneath the epithelium. The epithelium reached several times its normal thickness. Rats affected with otitis media rapidly accumulated purulent exudate in the tympanic cavity. Proliferative connective tissue eventually filled the tympanic cavities. The tympanic membranes also became markedly thickened due to proliferation of epithelial cells and connective tissue. The rhinitis produced the same morphological changes as described previously by Giddens et al. (1971a). Methods of Control and Prevention Nelson (1967) reported that attempts have been made to control CRD by biological means and by chemotherapy. He (Nelson, 1951) established a colony of CRD-free rats from parent stock by cesarean-section delivery and foster suckling the young on germfree mothers. Chronic respiratory disease, which had been present in the original colony, was eliminated in the cesarean-derived colony. The CRD-free colony and subsequent generations were maintained in rigidly isolated quarters. Outside rats were not introduced into the colony. Nelson (1967) stated that this colony was kept for several years in a CRD-free state before being discontinued. Foster (1958) developed a commercial CRD-free colony beginning with cesarean-section delivered pups and foster-nursed by germfree mothers. The CRD colony was maintained within barrier facilities that prevented the introduction of microbial pathogens. Many CRD- free rat colonies have since been developed utilizing this general protocol. 12 Kappel et a1. (1974) outlined steps to be followed for establish- ment and maintenance of a large-scale commercial rat breeding colony free from mycoplasmas. Defined flora foster parents without myco- plasmal unuaof infection in germfree isolatOrs were used to raise young derived by cesarean section from M; pulmonis-infected conven- tional donors. The offspring, who were free of mycoplasmas, were used to establish a breeding nucleus within a smaller barrier room. The breeding nucleus was in turn used to establish a production colony which was housed in a second larger barrier room. Colonies were monitored by routine diagnostic methods and by nasal washing techniques. Kappel et a1. (1974) further reported that previous efforts to rid laboratory rats of mycoplasmal microorganisms by therapeutic or prophylactic means were generally not successful. Haberman et a1. (1963) eliminated CRD in rats by oral administration of sulfa- merazine at the rate of 5 mg/20 gm of feed. Respiratory disease was eliminated in the third through tenth generations. Chlortetra- cycline given orally at the same rate did not eliminate respiratory disease. Gannaway and Allen (1969), at the National Institutes of Health (NIH), housed rats in filtered-air cages and gave them sulfamerazine in the drinking water at a rate of l gm/gallon of water. They concluded that the sulfamerazine prophylaxis was responsible for eliminating respiratory disease. In contrast to the findings at NIH, Lindsey et a1. (1971) reported that rats exposed to CRD and given sulfamerazine at a rate of 0.25 mg/ml of water developed severe rhinitis, otitis media, and lung lesions. However, when tetracycline was given at the rate of 0.25 mg/ml, the rats 13 failed to develop rhinitis or otitis media and only an occasional rat had suggestive hyperplasia of lymphoid tissue. Lamb (1975) reported that early histological screening, sampling of animals of all ages at frequent intervals, and further research into the problem of respiratory disease are necessary to provide ultimate solutions to the control of CRD in rats. Immune Response of Rats to Mycoplasma pulmonis Little is known about the immune response of rats to M. pulmonis. Lemecke (1961), at the University of Cambridge, demonstrated that in the early stages of the natural disease, when the organisms were confined to the nasopharynx, complement-fixing (CF) antibody values were very low. AS the disease progressed and the organisms invaded the lungs, circulating antibody titers increased. Whittlestone et al. (1972), working in the same laboratory as Lemecke, reported a correlation between the extent of pneumonia in rats and CF titers and pointed out that CF antibody may be a helpful indicator in determining M. pulmonis infection in rats. (They, however, found that both M. pneumoniae and M. pulmonis organisms persisted in the presence of high titers of cirulating CF antibodies, therefore suggesting that circulating antibody does not play an important role in protection or recovery from mycoplasmal infection. Kohn and Kirk (1969) detected plate agglutination titers in rats inoculated with M. pulmonis and in rats exposed indirectly to infected animals. Attempts by these investigators to demonstrate antibody titers by the metabolic inhibition test were essentially negative. 14 Cassell et a1. (1973) reported that rats inoculated intranasally with tracheal aspirates and tested 4 weeks later developed a positive delayed hypersensitive skin reaction following intradermal administra— tion of washed M. pulmonis cells. The skin reaction was characterized by infiltration into the dermis of lymphocytes, macrophages, and a few plasma cells. The mere demonstration of hypersensitivity did not prove its importance in the pathology of the disease or its role in protection against the disease. Therefore, it is apparent that further studies are needed to determine the role of hypersensitivity in M. pulmonis infections in the rat. Germfree Principles Pleasants (1974), at the Lobund Laboratory at the University of Notre Dame, has extensive1y reviewed the general literature on germ- free methodology. Trexler and Reynolds (1957) and Trexler (1959) reported that plastic germfree isolator units offer general advan- tages over previously used rigid materials. Transparency, flexibility of the units, lack of excess weight, multiplicity of sizes and shapes, inexpensiveness, and the fact that plastic units are partially sus- tained by enclosed air pressure were the primary advantages cited. The major disadvantage described was the possibility of puncture. Newton (1965), at the National Institutes of Health, reported that the basic germfree system consists of a sterile airtight struc— ture to house the experimental animals and methods for passing air, water, food, bedding, cages, and other needed supplies in and out of the isolator through some sterilizing device. He also mentioned that 15 plastic isolators are sterilized by chemicals usually applied directly to disinfect both the air Space and the surface of the unit. Peracetic acid in a 2% aqueous solution is the most widely used disinfectant for plastic isolators. Jaworski and Miller (1963) developed supply cylinders which can be autoclaved and then attached to the plastic unit for delivery of materials by way of a germicide connection. Sacquet (1968), in France, outlined a specific set of procedures for maintaining germfree animals. The importance of carefully following the principles for sterilization and keeping positive air pressure to prevent entry of microorganisms from the outside were strongly emphasized. Fuller (1968), in England, described procedures to follow when testing isolators for sterility. In routine control, materials from the isolator must be cultured and examined microscopically for evi- dence of contamination. Pleasants (1974) reported further that persons contemplating the use of germfree animals for research should consider the importance of the microbial variable to their study and the technical require- ments for the research. Technical factors outlined included: (1) availability of required animal species and strains, (2) availa- bility of animals to be used as controls, (3) technical skills required to complete the research, (4) availability of isolators and accessory equipment to maintain the animals, (5) interior equipment needed, (6) air supply and filters, (7) diets for the germfree animals, (8) water and bedding to be used, (9) sterilization of the isolators and supplies, (10) husbandry, and (11) microbial monitoring. 16 The rat is one of the many animals available for germfree research. Sprague-Dawley, Wistar, and Fischer strains of rats at Lobund have been extensively tested and reported to have no bacterial, fungal, protozoan, or viral contamination (Pellard and Kajima, 1967). Newton (1965) reported that weight gains of germfree rats compare favorably with those of conventional rats but that reproduction has been somewhat erratic. Infertility and ignoring, eating, or inability to nurse the young have been difficulties encountered. Dietary deficiencies due to oversterilization, too much inbreeding, and excessive noise or other disturbances may be contributing factors to poor reproduction. Summary of Literature Review The information in this literature review indicates that CRD is an important disease that complicates research using the rat. The clinical signs and gross and microscopic lesions characteristic of the natural disease are fairly well established. {Mycoplasma pulmonis is believed to be the primary etiologic agent of CRD, but additional information is needed as to the nature and pathology of the disease, especially factors that enhance or prevent infection. Germfree rats can be utilized for these studies, since they are free of mycoplasmas and other known pathogenic agents and therefore supply information uncomplicated by microbial contamination. OBJECTIVES The objectives of this research were: 1. To reproduce Myc0p1asma pulmonis infection in the rat with a known agent isolated from rats with the disease. 2. To reisolate and identify the agent producing the disease. 3. To compare the susceptibility of conventional, exgermfree, and germfree rats of different ages. 4. To determine the role, if any, of ammonia fumes in suscep- tibility to M. pulmonis infection. 5. To continue previous research started in this department on the pathogenesis, etiology and general nature of M. pulmonis infection in rats used in research. 17 MATERIALS AND METHODS This research was initiated in September 1975. It was conducted in the Pathology Laboratories, Barn 5, at the Veterinary Research Farm, and at the Clinical Microbiology Laboratory. Sources and Care of Experimental Animals Exgermfree (born and reared germfree and later changed to a conventional environment), conventional (reared in open room condi- tions in association with other rats of the same type), and germfree (free from demonstrable forms of associated life) rats were used for this research. The exgermfree rats were obtained from the Lobund Laboratories, University of Notre Dame. The conventional rats were first and second generation offspring of the original exgermfree rats. The germfree rats were obtained as weanlings from.a commercial source (A. R. Schmidt Co., Inc., Madison, WI). All rats were of the Sprague-Dawley strain. The exgermfree rats were maintained in filter-topped "shoebox" cages, initially containing up to 6 rats each. Male and female rats were separated except during breeding. The conventional rats were maintained similarly to the exgermfree rats except that air filter-topped cages were not used. The germfree rats were kept in flexible plastic isolator units measuring 24 x 24 x 60 inches (Trexler, 1959). Standard germfree procedures were 18 19 followed (Sacquet, 1968). Autoclavable, plastic "shoebox" cages fitted with stainless steel wire covers housed the rats within the isolator. The exgenmfree and conventional rats were fed a commercially prepared pelleted ration (Peerless Laboratory Diet, Peerless Pet Foods of Battle Creek, Inc., Battle Creek, MI) formulated for labora- tory rats and mice. Originally the germfree rats were fed the same ration (autoclaved) as the other rats, but later they were fed a commercially formulated autoclavable ration (Tekled Mouse/Rat Autoclavable Diet [L485], Mogul Corporation, Winfield, IA) when nutritional deficiencies developed due to sterilizing the diet. The cages were cleaned at weekly intervals. The germfree isolators were entered approximately 3 times monthly to replenish food, water, and litter and to remove waste material. Isolators were tested periodically for sterility according to the method of Fuller (1968). Water, litter, and feces were collected in screw-top tubes, removed from the isolator, and taken to the Pathology Labora- tories for cultures and microscopic examination. When germfree rats were necropsied, intestinal contents were cultured to further measure their microbial status. Necropsy and Tissue Preparation Procedures Rats were killed with an overdose of sodium pentobarbital given intraperitoneally. The skin was then reflected from the abdomen, thorax, and ventral cervical region. The trachea was located and severed at the laryngotracheal junction. Material for culture was collected from the trachea and lungs by tying a cannula securely into 20 and trachea and injecting 2.0 ml of.Mgcoplasma medium into the lung and withdrawing a minimum of 0.5 mm back into the syringe. The lung perfusion technique has worked well because mycoplasmal microorganisms are located almost entirely on the surface of the bronchial epithelial cells (Taylor-Robinson et al., 1972). Next, the thoracic cavity was opened and the trachea and lungs were removed intact. Enough fixative was infused into the tracheal cannula to slightly distend the lungs. The trachea and lungs were fixed in 10% buffered formalin. The skin was reflected from the head, and 0.4 ml of broth was injected and withdrawn from the tympanic cavity of the right ear. The head was removed at the atlanto-occipital joint and placed in formalin solution for later decalcification. The intact heads were demineralized in R.D.0. solution (DuPage Kinetic Laboratories, Inc., Downers Grove, IL). Sectioning of the heads included a transverse section midway between the external nares and the medial canthi, and a transverse section of the middle ear through the external auditory meatus. Selected tissues for microsc0pic examination were paraffin embedded, cut at 6 microns thickness, and stained with hematoxylin and eosin. Techniques used were essentially those suggested by the Armed Forces Institute of Pathology (Luna, 1968). Experimental Exposure of the Rats The exgermfree and conventional rats were exposed to M. pulmonis by aerosol inhalation. Fourth passage, 72-hour broth culture was used. A total of 5 ml was given per cage of 5 or 6 rats. The aerosol was placed in a glass nebulizer (Model 640 Nebulizer, The DeVilbiSS 21 Co., Toledo, OH) using a low pressure stream of compressed nitrogen gas. The spray was directed through a hole cut in the filter top cage caps. After all of the broth was nebulized, the hole in the filter cap was sealed with plastic tape to trap the mist inside the cages. To expose the germfree rats, screw-cap vials containing 5 m1 of M3 pulmonis in broth culture were first washed in 70% ethyl alcohol and then passed into the germfree isolator through the entry lock. The peracetic acid fumes were allowed to dissipate from the isolator for 45 minutes, after which the isolator's air inlet and outlet were sealed. A portion of the broth culture was applied to the nostril area of the rats using a.l ml tuberculin syringe. The remaining broth culture was placed on the feed and bedding within the cages. Micrdbiologic Techniques Mycoplasma pulmonis strain M/J 69 (Jersey et al., 1973) was used in this research. The organisms had been stored at -64 C, were thawed and then passed 3 times in exgermfree rats (experiment 1) to enhance their virulence. The media used for isolation and purification of M. pulmonis and for preparation of inoculation material was constituted from commercially purchased dehydrated products as suggested by the suppliers. Broth (Bacto-PPLO Enrichment Broth, Difco Laboratories, Detroit, MI) and agar (Bacto-PPLO—Agar, Difco Laboratories, Detroit, MI) media were supplemented with horse serum, yeast extract and inhibitory levels of thallium acetate and penicillin to retard bacterial growth (BBL Mycoplasma Enrichment 11865, Becton, Dickinson, and Co., Cockeysville, MD). The general procedures followed were 22 essentially those of Jersey et a1. (1973). Mycoplasma agar was plated with 0.1 m1 broth from the original 72-hour cultures, and the plates were checked at 3 and 6 days. MyCOplasma pulmonis was purified generally as outlined by Whittlestone et al. (1972). Verification of the inoculum as M. pul- monis was made by the growth inhibition (GI) test as described by Clyde (1964) and later modified by Stanbridge and Hayflick (1967). In this test anti-M. pulmonis and antisM. pneumoniae sera.were pur- chased from a commercial laboratory (Microbiological Associates, Bethesda, MD) and used for identification. Mycoplasma pulmonis was identified on the basis of an area of reduced growth surrounding a paper disc impregnated with the specific antiserum. The M. pneu- moniae antiserum produced no such inhibition. Experiments Sequential experiments were conducted to reproduce CRD in rats, to identify the causative agent, and to determine the effect of ammonia fumes on the susceptibility to M. pulmonis infection. For convenience in presentation of data, some details of the procedure will be included in the results. Experiment 1 Fifteen of 25 originally obtained exgermfree rats, 3 to 5 months of age, were used to enhance the virulence of M. pulmonis M/J 69. The original culture had been frozen in vials for 2 years. Five rats were exposed at a time with the 3 exposures being approximately 1 month apart. The 10 additional rats in this group were used as controls. Two of the rats were killed and examined before the 23 experiment to be sure they were free from infection. Eight rats were maintained for the duration of the experiment as colony controls. Experiment 2 TWenty-two, 3-month-old, conventional rats were used to study the course of CRD infection. Two rats were killed and examined before the experiment to be sure they were free from infection. Twenty animals were placed, 5 per cage, in 4 filter-top boxes and exposed to M. pulmonis. They were killed at approximately weekly intervals starting 21 days following exposure. Experiment 3 Five, 3-month-old, conventional rats were used to study the long-term course of CRD infection. The animals were placed in a filter-top box and exposed to M; pulmonis. One rat died 162 days following exposure and the remaining rats were killed 190 days after exposure. Experiment 4 Thirty-eight conventionally reared rats approximately 45 days of age were used to evaluate the influence of ammonia fumes on the course of CRD. Two rats were killed and examined before the experi- ment to be sure they were free from infection. Thirty-six were placed, 6 per cage, in 6 filter-top boxes. Twelve rats were exposed to M. pulmonis only. The second group of 12 rats was exposed to M. pulmonis plus ammonium hydroxide in open bottles 1 inch in diameter. The final group of 12 rats was exposed to ammonium hydroxide only. 24 Experiment 5 Six germfree rats were used. Two were killed and examined before the experiment to make sure they were free of identifiable microorganisms. Four rats were placed in a single germfree isolator and inocur lated intranasally with a broth culture of M. pulmonis. Following inoculation the rats were housed in a single clear plastic "shoebox" cage. Rats were killed when severe dyspnea developed. The number and sex of the rats used in this experiment were determined by their availability at the time the experiment was conducted. Cology Controls Eight, exgermfree rats (from Experiment 1) were maintained in a separate room at Barn 5 throughout the duration of this research. This room also maintained a guinea pig colony. These rats were killed and examined for evidence of respiratory disease at the termination of the research. RESULTS Chronic respiratory disease was produced in the experimental rats. The susceptibility to infection, clinical nature of the disease, and lesions varied somewhat among the rats in the different experiments. These differences were apparently related to the age of the rats, the methods of rearing, the presence of amonia fumes, and other factors. The results of each experiment are tabulated (Table 1). A narrative summary of each experiment and a composite description of the clinical nature, pathology, and microbiology of the disease in the different experiments follows. Sumnary of Experiments Experiment 1 The results were similar in each consecutive passage. The clinical signs, gross lesions and.microscopic lesions were those described later in the composite description of CRD. The first clinical signs were noticed 10 days after exposure to M. pulmonis. The gross lung changes were most often diffuse, and the apical and dependent portions of the lungs were frequently affected. Histo- logic lesions were present in the lung, nasal cavity, and middle ear tissues from all of the rats. Tracheitis was observed in 14 of the 15 rats. 25 Table 1. Experimental design, 26 lesions and Mycoplasma pulmonis isolations Killed or died of Experiment Age ** Sex exposure Number Treatment No. (mo.) M F Rearing (days) 1 M. pulmonis 15 3-5 15 - Exgermfree 14-49 Controls 2 3 2 - Exgermfree --- 2 M. pulmonis 20 3 10 10 Conventional 21-50 Controls 2 3 l 1 Conventional --- 3 M; pulmonis 5 3 5 - Conventional 162-190 4 NH3 12 1.5 6 6 Conventional 20-57 M. pulmonis 12 1.5 6 6 Conventional 20-57 M. pulmonis 12 1.5 6 6 Conventional 20-57 + NH 3 Controls 2 1.5 1 1 Conventional --- 5 M. pulmonis 4 3 - 4 Germfree 10-41 Controls 2 3 - 2 Germfree --- * CC Controls 8 12 2 6 Exgermfree --- * Colony controls. * * Age exposed or killed if not exposed. ~- 27 Site and Frequency of M. pulmonis Isolations Trachea- Site and Frequency of Lesions Microscopic Lesions Gross Lesions Nasal Middle bronchial Middle Lungs Lungs Trachea cavity ear washings ear 10/15 12/15 14/15 15/15 15/15 7/15 9/15 0/2 0/2 0/2 0/2 0/2 0/2 0/2 17/20 18/20 20/20 20/20 20/20 8/20 13/20 0/2 0/2 0/2 0/2 0/2 0/2 0/2 5/5 4/5 5/5 5/5 5/5 2/4 2/4 0/12 0/12 12/12 12/12 12/12 0/12 0/12 1/12 6/12 12/12 12/12 12/12 5/12 1/12 2/12 7/12 12/12 12/12 12/12 4/12 1/12 0/2 0/2 0/2 0/2 0/2 0/2 0/2 4/4 4/4 4/4 4/4 3/4 4/4 2/4 0/2 0/2 0/2 0/2 0/2 0/2 0/2 3/8 4/8 5/8 6/8 4/8 3/8 3/8 28 Mycoplasmas*were isolated from the tracheobronchial washings from 7 of the 15 exposed rats. Overgrowth of bacteria on the agar media from the washings of the remaining 8 rats was present. Bac- terial colonies were noted on the media in 3 of the plates on which mycoplasmas were identified. Mycoplasmal microorganisms were iso- lated from middle ear cultures from 9 of the 15 rats. Four addi- tional plates were overgrown with bacteria, and 2 were without mycoplasmal or bacterial growth. No clinical signs or lesions of the respiratory tract and middle ear were present in the 2 control rats. Mycoplasmas were not isolated from the rats, but bacterial colonies grew from the tracheobronchial washings from 1 rat. Experiment 2 Clinical Signs, gross lesions, and microscopic lesions were essentially those described later. The onset of clinical signs was noticed 12 days after exposure to M. pulmonis. Gross lung lesions, observed in 17 of the 20 rats, were generally diffuse and affected all portions of the lungs. Microscopic lung lesions were present in lungs of 18 of the 20 rats exposed to M. pulmonis. Tracheitis, rhinitis, and otitis media were present in all of the rats. Mycoplasmas were isolated from tracheobronchial washings of 8 of the 20 exposed rats. Overgrowth of bacteria was present from the washings of 9 rats and no microorganisms were isolated from washings of 3 rats. Mycoplasmal microorganisms were identified from middle ear cultures of 13 of the 20 exposed rats. Bacterial overgrowth 29 was observed in cultures from 4 rats. Results from the middle ear cultures from the remaining 4 rats were negative. No clinical signs or respiratory tract and middle ear lesions were present in the 2 control rats killed prior to the beginning of the experiment. Mycoplasmas were not isolated from these rats. Bacterial growth occurred from the tracheobronchial washings, but not from the middle ear cultures. Experiment 3 The clinical signs, gross lesions, and microscopic lesions were basically those described later. The onset of clinical signs was as described in Experiment 2. Except for the rat that died, all rats developed relatively mild clinical signs which persisted throughout the experiment. Gross and microscopic lesions were seen in the lung tissues from 4 of the 5 rats. The lung lesions were both focal and diffuse, and areas resembling abscesses were noted on the lung sur- face of 2 rats. Middle ear, tracheal, and nasal cavity lesions were observed in tissues from all 5 rats. Mycoplasmal microorganisms were isolated from the tracheobronchial washings of 2 of the 4 rats cultured and from the middle ears of the same 2 rats. Bacterial growth was present on the media in all of the plates examined. The fifth rat in the experiment died during the weekend and microbiological work was not done. Experiment 4 Lesions of the middle ear, nasal cavity, and trachea were present in the 12 rats exposed to only M. pulmonis. The lung changes, when present, were generally a marked lymphoid hyperplasia around large 30 bronchi. In 1 rat a small solid lesion was observed and the lung changes were more diffuse than in the other rats. Mycoplasmas were recovered from the tracheobronchial washings of 5 of the 12 rats. Bacteria overgrew the media in the remaining 2 plates. Mycoplasmal organisms were isolated from.the middle ear of l of the 12 rats. Cultures from the remaining rats were overgrown with bacteria. Tracheitis, rhinitis, and otitis media were observed in the 12 rats exposed to M. pulmonis plus ammonia fumes. The tracheitis and rhinitis were more severe than noted in those rats exposed to M. pulmonis only. The lung changes, which were observed in 3 of the 12 rats, resembled those in the rats exposed to only M. pulmonis. Mycoplasmas were recovered from the tracheobronchial washings of 4 of the 12 rats. Bacterial overgrowth was noted from the washings in an additional 6 rats, and tracheobronchial cultures from the remaining 2 rats were negative. Mycoplasmas were isolated from the middle ear of l of the 12 rats. Six middle ear cultures were over- grown with bacteria, and no growth was noted on the media in the remaining 5 plates. The 12 rats exposed to ammonia fumes alone developed a tracheitis, an otitis media, and a severe rhinitis. A slight lymphocytic infil- tration of questionable significance was noticed around the lung bronchi in 4 of the 12 rats. Mycoplasmas were not isolated from tracheobronchial or middle ear washings from these rats. Bacterial overgrowth was noted on the media in several of the plates. No clinical signs, respiratory tract lesions, or middle ear lesions were noted in the 2 rats killed prior to the beginning of the experiment. Mycoplasmas were not isolated from the respiratory 31 tract or middle ear of these rats. Bacterial overgrowth was observed on the media in 1 plate cultured from the middle ear. Experiment 5 The 4 rats given an intranasal inoculation of M. pulmonis developed dyspnea and became depressed in 9 to 24 days. Diffuse lung lesions, tracheitis, rhinitis, and otitis media were observed in the 4 exposed rats. The changes are described later. Myco- plasmas were recovered from the trachedbronchial washings from all exposed germfree rats and from the middle ear cavities of 2 of the 4 rats. No growth of bacteria was present. The 2 rats killed before the experiment were free of identi- fiable microorganisms. No lesions suggestive of CRD were seen in the lung tissue, bronchi, trachea, nasal cavity, or middle ear of either rat. Colony7Controls All of the rats appeared to be healthy at the end of the experiments, except that 2 of the 8 rats were smaller than the others. Gross lung lesions were present in 3 of the 8 rats, and microsc0pic lesions were present in 4 of the 8 rats. Tracheitis was observed in 6 of the 8 rats, but the inflammation was mild. A mild rhinitis was present in 6 of the 8 rats. Middle ear involve- ment was present in 4 of the 8 rats. The lesions were of the nature of those described later. Mycoplasmal microorganisms were isolated from the tracheobron- chial washings of 3 of the 8 rats. Bacterial overgrowth was present on the media plates from 4 additional rats. One plate was negative 32 microbial growth. Mycoplasmas were isolated from the middle ear of 2 rats. The plates from the middle ears of 6 rats were overgrown with bacteria. Composite Description of CRD Clinical Signs Clinical signs were not readily apparent in many of the exgerm- free or conventional rats exposed to M; pulmonis. It was generally not possible to predict the severity of respiratory lesions based on clinical signs. The clinical Signs tended to be variable among rats even within individual cages. The first clinical signs appeared within 3 weeks after exposure. An infrequent sniffling and rubbing the nose and face with the front paws were noted. This sneezing was accompanied by a general depres— sion, fast respirations, and the appearance of a dirty hair coat. Many of the 45-day-old rats (Experiment 4) exposed to only M. pul- monis developed these signs and later appeared to recover. The more pronounced clinical Signs, when present, were a stunted appearance, labored breathing, reduced food consumption, and arching of the back (Figure 1). Terminal signs were gasping with the mouth held open and the neck stretched forward and a tendency to huddle in the corners of the cages. These clinical signs were observed in 3 of the germfree rats (Experiment 5) and 2 other rats that died (Experi- ments 1 and 2). All rats that were exposed to ammonia fumes almost immediately developed excessive tearing, sniffling, and gasping. These signs generally subsided within 2 to 3 days. 33 Figure 1. Clinical Signs of CRD in a 55-day-old rat reared germfree, 25 days after exposure to a broth culture of M. pulmonis. Although rough hair coat, emaciation, and dyspnea were evident in this rat, only an arched back and rubbing of the nose and face with the front paws are clearly recognizable in the photograph. Figure 2. Dorsal view of lungs in a 9-month-old, con- ventionally reared rat, 6 months after aerosol exposure to M. pulmonis. Note the demarcation between the solidified por- tions (arrows) and the normal portions of the lungs. Mucopurulent exudate was draining from the trachea but is not discernible in the photograph. 34 Figure l Figure 2 35 Gross Lesions The major gross changes were in the lungs (Figure 2) and the trachea. The affected lung tissue was usually sunken, firm, and solid. The solidified (consolidated) areas were dark red initially, later becoming mottled with reddish, grayish, and yellowish areas. The lung solidification was accompanied by the production of a yellowish slimy exudate and dilatation of the bronchi. Focal lesions resembling abscesses were present in some of the lungs. The lung lesions involved 5 to 60% of the total lung mass and affected portions of lung lobes or entire lobes. More than 1 lung lobe was usually involved. Muc0purulent exudate was frequently observed in the tracheal lumen. The amount was slight to excessive. The tympanic membrane was distended in an occasional rat, due to the accumulation of purulent material in the middle ear. Microscopic Lesions Histopathologic changes were observed in the lung tissue, the bronchial tubes, the trachea, the nasal cavity, and the middle ear. Significant bronchial changes were dilatation of the bronchi (bron- chiectasis) and proliferation (hyperplasia) of the bronchial epithelium. Other alterations were an epithelial invasion of neutrophils, coagula- tion necrosis of the bronchial epithelium, and an accumulation of mucus and pus with many neutrophils in the bronchial lumen. This exudate occluded the lumen in many sections. Changes around the bronchi and bronchioles were evident. Peribron— chial and peribronchiolar lymphocytic infiltration of varying degrees t d g“ Figure 3. Bronchiole and surrounding tissue of a 3-month- old, exgermfree rat. Note the lymphoid tissue in the wall of the bronchiole (arrow), the lumen of the bronchiole (L), and a blood vessel filled with erythrocytes (V). The breaking off of the alveolar walls was likely from infiltrating the lung with media and/or 10% formalin solution. B a E stain; x 312. Figure 4. Subacute bronchiolitis showing peribronchiolar lymphocytic infiltration in a 4-month-old, conventionally reared rat, 29 days after exposure to M. pulmonis. Note the lumen (L), thickened, hyperplastic bronchiolar wall (W), partial collapse of adjacent alveoli (A), and collection of lymphoid cells (C). Atelectasis was also observed surrounding the bronchiole. H a E stain; x 125. 37 Figure 3 Figure 4 38 was noted. Similar changes were observed around blood vessels. In rats with a chronic infection, connective tissue proliferation was noted around the bronchi and bronchioles and in collapsed and solidi- fied (consolidated) areas of the lung. The degree of pulmonary involvement was variable, and portions of lung free from noticeable macroscopic changes were always present. The overall lung changes were those of bronchopneumonia (Figures 3,14 and 5). Subacute tracheitis (Figures 6 and 7) was a consistent finding. The tracheitis was characterized by a hyperplastic increase in the number of tracheal epithelial cells. In a few of the rats (especially those exposed to ammonia fumes) necrosis of the epithelial surface of the trachea was present. Cyst-like invaginations of the mucous membranes were observed. In areas of epithelial hyperplasia, the mucus-forming glands of the trachea were hyperplastic. The tracheitis was more severe in the rats exposed to ammonia fumes alone and ammonia fumes plus M. pulmonis than in those rats exposed to M. pulmonis alone. Subacute rhinitis (Figures 8, 9 and 10) with exudation into the nasal passage, infiltration of lymphocytes below the epithelium, and an increased number of goblet cells were the major changes observed in the nasal cavity. Necrosis of the nasal epithelial lining was seen in a few instances. The mucus-forming glands of the submucosa were hyperactive. Otitis media (Figures 11, 12 and 13) with an accumulation of purulent exudate within the tympanic cavity was present. Connective tissue proliferation and formation of cystic spaces were commonly 39 Figure 5. Bronchiolitis showing a respiratory bronchiole, dilated and filled with exudate, 40 days after exposure of an exgermfree rat to M. pulmonis. Note the purulent exudate with many neutrophils (E), the somewhat thickened epithelium (T), and accumulation of lymphocytes, macrophages, and neutrophils around the bronchiole (B). H a E stain; x 312. Figure 6. Tracheitis in a 95-day-old, conventionally reared rat exposed to M. pulmonis and ammonia fumes at 45 days of age. Note the loss of epithelium with a deep invagination (arrow), the mucinous degeneration of the glands of the lamina propria (D), the infiltration of lymphocytes and plasma cells into the lamina propria (L), and submucosa (S). H & E stain; x 125. 4O Figure 6 41 Figure 7. Higher magnification of Figure 6. Note the glandular degeneration and cellular infiltration into the lamina propria. H & E stain; x 312. Figure 8. Purulent rhinitis in an exgermfree rat that was exposed to M. pulmonis at 3 months of age and died 42 days later. Note the purulent exudate with neutrOphils in the lumen (E), the epithelium with an increased number of goblet cells (G), and the subepithelial infiltration of inflammatory cells (I). H & E stain; x 125. 42 Figure 7 Figure 8 43 Figure 9. Purulent rhinitis in a lOZ-day-old, conventionally reared rat that was exposed to M. pulmonis and ammonia fumes at 45 days of age. Note the purulent exudate in the nasal cavity (N), the epithelium (E), and the accumulation of inflamatory cells in the lamina propria (C). The space between the carti- laginous median septum (S) and the lamina propria is an artifact. H & E stain; x 125. Figure 10. Higher magnification of Figure 9. Note the neutrOphils which have infiltrated into the epithelium. H a E stain; x 312. ' 44 Figure 9 Figure 10 45 Figure 11. Otitis media in a 4-month-old rat that was reared germfree and exposed to M. pulmonis at 3 months of age. Note the large space filled with tympanic exudate (P), the smaller space filled with inflammatory cells and exudate (C), the tissue proliferation around the abscessation (T), and the osseous tympanic wall (0). H 5 E stain; x 50. Figure 12. Higher magnification of the tympanic cavity of the same ear as in Figure 11. Note the neutrOphils (N) and cystic space (S). H a E stain; x 125. 46 Figure 11 Figure 12 47 Figure 13. Higher magnification of tympanic cavity of same ear as Figures 11 and 12. Note the inflammatory cells and vacuolated areas. H a E stain; x 312. ‘1'... nllllli 48 observed. Alternate areas of connective tissue with cystic Spaces and purulent exudate were present in some of the rats examined. Inner ear involvement was not observed. Microbiology Isolation of mycoplasmal microorganisms by Sites and frequencies was discussed with each experiment. Colonies of M. pulmonis grown on agar medium were detected as early as the day after the plates were inoculated. The optimum time to examine the plates was 72 hours after inoculation. Although the plates were kept for a minimum of 1 week, little additional growth was evident after 72 hours. For most of the rats, bacterial cultures were not done in parallel with the cultures for mycoplasmal microorganisms. As emphasized in the discussion of each experiment, considerable over- growth of bacteria on the mycoplasma agar medium was present. This growth was often observable within 12 hours after inoculation. DISCUSSION The clinical nature and lesions of CRD were essentially as described by previous researchers (Giddens et al., l97la,b; Jersey et al., 1973). The lesions of the lungs have been given primary consideration in mudh of the previous research on CRD. In fact, Innes et a1. (1956) and Newberne et a1. (1961), in an examination of several hundred rats, failed to mention changes in the middle ear and upper respiratory tract. Rhinitis, tracheitis, and otitis media were more prevalent than were lung lesions in the exgermfree and conventional rats in my experiments. Therefore, I believe that investigators should give more attention to upper respiratory and middle ear lesions in research on CRD. Considerable difficulty was encountered in identifying M. pulmonis from tissues of the exgermfree and conventional rats because of bacterial overgrowth on the mycoplasma media plates. Rather than reliance on the inhibitors in the mycoplasma media, one might con- sider the possibilities of immunofluorescence and electron microscopy to identify M; pulmonis from the respiratory systems and middle ears of exgermfree and conventional rats. Mycoplasma pulmonis, on the other hand, was readily reisolated and identified from tracheo- bronchial washings from the germfree rats. The germfree rats were most susceptible to CRD infection. They developed a marked respiratory distress, and lesions of the 49 50 respiratory tract were extensive. An explanation for the increased susceptibility would be the lack of contact of the germfree rats with agents to stimulate their body defense mechanisms. The patho— logic changes in the exgermfree and older conventional rats were similar but developed more slowly than did the lesions in the germ- free rats. No appreciable differences in susceptibility to infec- tion or in the course of the disease were noticed when comparing the exgermfree and conventional rats of similar ages. This finding contradicted that of Kasali (1974), in which the exgermfree rats were more susceptible to M. pulmonis infection than‘were conventional rats. The younger conventional rats (Experiment 4) did not readily develop pulmonary lesions but did develop upper respiratory and middle ear lesions. Ammonia fumes did not enhance the suscepti- bility to M. pulmonis lung infection, but they did cause a more severe rhinitis and tracheitis. The ammonia fumes alone produced lesions in the trachea and nasal cavity closely simulating those of.M. pulmonis in germfree rats. The rats also unexpectedly developed otitis media. One explanation might be that the ammonia fumes made the rats more susceptible to an incidental bacterial infection. The accumulation of ammonia in animal cases from urine and the effects of this chemical on the respiratory system need to be considered in designing M. pulmonis experiments and also inhalant toxicity studies utilizing rats. Husbandry aspects should not be overlooked when designing respiratory experiments using rats. It is the experience of the author that many researchers do not give husbandry due consideration when planning their experiments. 51 The isolation of M. pulmonis and the presence of CRD lesions in the colony controls was not expected. The rats were maintained in a separate room for 9 months and did not have direct contact with other diseased rats. No clinical signs were noted during the 9 months. The infection could have originated from infected rats in an adjacent room, contamination in husbandry, wild rodent carriers, or the guinea pigs housed in the same room. Judging from the increased susceptibility of the germfree rats, the development of more uniform clinical signs and lesions,and the lack of exogenous contamination, the germfree rat is a suitable model for studying M. pulmonis infection. Germfree rats may also be a useful model to conduct research on the role of mycoplasmal infections in a variety of other species, including man, and thus be of value in comparative medicine. SUMMARY Research was conducted to reproduce Myc0p1asma pulmonis infec- tion in rats reared conventionally, exgermfree, and germfree. Ninety- six rats were used in 5 sequential experiments. Chronic respiratory disease was reproduced in rats exposed to broth cultures of M; pulmonis. The clinical signs and lesions were characteristic of the natural disease. Clinical signs were observed in the rats by 3 weeks after exposure to M. pulmonis. The most severe clinical signs were seen in the germfree rats. These rats developed dyspnea and became depressed. The young conventionally reared rats, exposed to M. pulmonis at 45 days of age, developed only mild clinical signs. The mild signs were often followed by apparent recovery. Tracheitis, rhinitis, and otitis media were present in rats exposed to M; pulmonis regardless of age and method of rearing. The tracheitis and rhinitis were most severe in rats exposed at 45 days of age to both M; pulmonis and ammonia fumes. Also, upper respiratory lesions and purulent exudate in the middle ear were observed in the conventional rats exposed to only ammonia fumes. Bronchopneumonia with pulmonay solidification (consolidation) was a consistent finding in conventionally reared, exgermfree, and germfree rats exposed to M. pulmonis at 3 months of age or older. Conventional raised rats exposed to M. pulmonis alone and to M. 52 53 pulmonis and ammonia fumes at 45 days of age developed fewer lung lesions. Upper respiratory and middle ear changes but no significant lung lesions were observed following exposure of 45-day-old conven- tional rats to ammonia fumes only. Mycoplasma pulmonis microorganisms were isolated from pre- viously exposed rats. The reisolation procedures were complicated by the presence of contaminating microorganisms in the conventional and exgermfree rats. Chronic respiratory disease in the rats was primarily due to M. pulmonis. Ammonia fumes and contaminating microorganisms may con- tribute to the disease as it occurs naturally in rat colonies used in research. REFERENCES REFERENCES Brennan, P. C., Fritz, T. E., and Flynn, R. 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In The Veterinary Annual, 15th Issue (ed. by Grunsell, C. S. G., and Hill, F. W. G.). John Wright and Sons, Ltd., Bristol, England, (1975). VI TA The author was born in Auburn, Indiana, on January 13, 1940. He received his primary and secondary education in the Eaton, Indiana, Public Schools. After graduation from high school in 1958, he continued his education at Purdue University, Lafayette, Indiana, and received a Bachelor of Science Degree in Agriculture in August 1962. He taught general soience at Woodview Junior High SChool in Indianapolis for one year. In September 1963, the author reentered Purdue University and was awarded a Doctor of Veterinary Medicine Degree in June 1968. In the sumers of 1962 through 1966, he attended Ball State University and was awarded the Master of Arts Degree in August of 1966. From September 1968 to June 1973, the author held an appoint- ment as an Assistant Professor in the Department of Physiology and Health Science at Ball State University and was engaged in part-time private small animal practice in Muncie, Indiana. From June 1973 to September 1974, he held an appointment as a resident instructor with the Center for Laboratory Animal Resources at Michigan State University. Following a brief period in small animal practice in South Bend, Indiana, the author returned 58 59 to Michigan State University in September 1975 in the Department of Pathology as a Master's Degree candidate. The author married Miss Carol Judy Brault in 1968. GAIN STAIE UNIVERSITY LIBRHARIES ImIgII IIII III III LII; lj