Br. J. exp. Path (1976) 57, 339
PATHOGENESIS AND PATHOLOGY OF RESPIRATORY TULARAEMIA IN THE RABBIT A. BASKERVILLE AND P. HAMBLETON Frcomtt the Mlicrobiological Researcn Establishment, Porton, Salisbury, W'Viltshire Received for publication January 27, 1976
Summary.-The development of pathological lesions in the organs of rabbits was examined at intervals from 1 h to 4 days after aerosol infection with Francisella tularensis. The earliest change, accumulation of polymorphonuclear leucocytes (PMN) in pulmonary alveolar ducts, occurred at 19 h. From the 2nd day multiple foci of necrosis and PMN infiltration were present in large airways and alveoli throughout the lungs and progressively increased in size. Pulmonary arteritis was a prominent feature of the infection. Areas of necrosis were present in the nasal mucosa, pharynx and trachea, and pyogranolomatous lesions consistently developed in the liver, spleen, and lymph nodes.
Tularaemia, caused by the Gramnegative bacterium Francisella tularensis, is an important zoonosis in many parts of the world. The disease occurs in a large number of feral and domestic animals and one of the most important hosts which serves as a source of human infection is the rabbit (Jellison and Parker, 1945). Epizootics of tularaemia develop not infrequently in rabbits, hares and rodents in Europe, Russia and North America, and each year there are a number of fatal human cases associated with such outbreaks. Since the rabbit is a natural host for F. tularensis this species was used as the experimental animal in an investigation of the early clinico-pathological and histopathological changes which take place in tularaemia, and of the modification of the disease bv vaccination. Inhalation of F. tularensis in aerosols is thought to be one of the commonest modes of human infection, and this frequently results in pneumonia (McCrumb, 1961). To simulate this route of infection as closely as possible susceptible rabbits were exposed to aerosols of virulent F. tularensis to provide basic information for subsequent studies on animals which had been vaccinated
against tularaemia and challenged with virulent organisms. The pathology of fatal tularaemia in man and of the experimental disease in rodents and monkeys has been described by a number of workers since the disease was first recognized in the early years of this century (Lillie and Francis, 1937a, b; Blackford and Casey, 1941; Downs et al., 1947; White et al., 1964; Landay et al., 1968). Published information on the pathology of tularaemia in the rabbit is confined to the terminal condition following subcutaneous inoculation of the organism (Lillie and Francis, 1937a) and to the peracute disease induced by a vaccine strain of F. tularensis injected i.v. (Finegold et al., 1969). This communication describes the sequential histopathological changes occurring in the organs of rabbits infected by an aerosol containing the virulent Schu-S4 (Eigelsbach, Braum and Herring, 1951) strain of F. tularensis. MATERIALS AND METHODS
Micro-organism-Francisella tularensis, virulent Schu-S4 strain (Eigelsbach et al., 1951) was used. Growth of bacteria and preparation of washed
340
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suspensions.-Bacteria were grown in batch culture in the medium of Sharer, Klein and Lincoln (1968), harvested and washed twice with 1% (w/v) peptone + 0-1% (w/v) cysteine HC1 (cysteine broth) and resuspended in cysteine broth as described by Hambleton et al. (1974). For aerosolization bacteria were resuspended in cysteine broth containing antifoam agent (Strange et al., 1972). Determination of numbers of viable bacteria.Numbers of viable F. tularen8si in suspensions of bacteria or tissue homogenates were determined as described by Strange et al. (1972). Animals.-Seventeen New Zealand White male and female rabbits were used. Infection of animals with F. tularensis.Aerosols of F. tularensis Schu-S4 generated with a 3-jet Collision spray from suspensions containing 5 x 109 or 2 x 1011 viable bacteria/ml were held in a Henderson (1952) apparatus and sampled for 1 min with a Porton Raised Impinger (May and Harper, 1957) having a flow rate of 11 I/min and containing 10 ml of cysteine broth + antifoam agent. Thirteen rabbits were infected by exposure for 1 min to bacterial aerosols containing 2 x 105 or 4 x 108 viable bacteria/l. The calculated inhaled retention doses of viable bacteria given to 10 rabbits were 1-2 x 105andtothe3others2-4 x 108. Four rabbits served as uninfected controls. Necropsy stages and procedure.-Preliminary studies showed that in the rabbit the disease runs an acute course and usually proves fatal after 4 or 5 days. Rabbits were therefore killed during the first 4 days after infection. The 3 given a dose of 2-4 x 108 organisms were killed, 1, 5 and 10 h after exposure, and of the animals given 1-2 x 105 bacteria 2 were sacrificed at each of the following stages: 19 h, 24 h, 2, 3 and 4 days. One control rabbit was killed each day, from the first to the 4th day. All animals were killed by i.v. or i.p. injection of pentobarbitone sodium. At necropsy carried out immediately after death the following organs were removed and portions fixed in 10% formol saline: ventral nasal turbinate bones, soft palate and palatine tonsils, larynx, trachea, lungs, heart, liver, spleen, kidneys, small intestine and brain. The cervical, bronchial and mesenteric lymph nodes were fixed for 1 h in Carnoy's fluid and subsequently transferred to 80% alcohol. The larynx and turbinate bones were decalcified in modified Gooding and Stewart's fluid. All tissues were processed by standard methods and embedded in paraffin wax. Sections cut at 5 ,um were stained with haematoxylin and eosin. Selected sections were stained with Mallory's phosphotungstic acid haematoxylin, by Gordon and Sweet's method for reticulin, with methyl green-pyronin, by periodic acid-Schiff, by Gram's stain, and by the combined Verhoeff's elastin and Van Gieson methods.
Bacteriological examination of homogenized tis8Ues.-Portions of organs excised at necropsy from infected rabbits were homogenized in a small volume (about 3 ml) of cysteine broth and viable F. tularensis detected as described above. Bacterial isolates were confirmed as F. tularensis on the basis of colony morphology, Gram staining and agglutination by rabbit anti-F. tularensis serum. RESULTS
Clinically the animals were dull and had slightly elevated temperatures by the 2nd day. On the 3rd day food intake was greatly reduced and there was diarrhoea and pyrexia in excess of 40.50. Pathological changes were not detected in the lungs or other organs of the rabbits killed at 1, 5 and 10 h after infection. For the other animals, changes were not found in organs not specified in the following description. 19 h
Lungs.-The earliest recognizable lesion developed at this stage and consisted of a small number of peripherally situated foci of polymorphonuclear leucocytes (PMN), principally in the region of alveolar ducts or affecting a group of several alveoli. F. tularensis organisms could not be found in these foci or at other sites in Gram-stained sections, probably because of their extremely small size and similar staining properties to those of the tissue, though the organism was isolated consistently from the lungs at all stages of infection from 19 h onwards. Liver.-In one animal only, the liver contained a few small accumulations of PMN. These had no consistent lobular distribution and there was no evidence of damage to hepatic cells. 24 h
Lungs.-Numerous small foci of PMN were scattered peripherally and involved alveolar ducts and alveoli (Fig. 1). Alveolar capillaries in affected regions were intensely congested. In a number of large bronchi and bronchioli there was localized infiltration of the epithelium and lamina propria by PMN. Bacteria could not be
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341
FIG. 1. Typical focus of PMN infiltration and necrosis in lung at 24 h. H. and E. x 120. FIG. 2.-2 days after infection. Focal necrosis of bronchial epithelium extends into the surrounding tissue. H. and E. x 50. FIG. 3.-Pulmonary arteritis on 2nd day. There is cellular infiltration accompanied by gross distortion of the endothelial cell layer. H. and E. x 110. FIG. 4.-Higher magnification of Fig. 3. Inflammatory cells have accumulated beneath the endothelium, which is disrupted at several points, and are invading the media. H. and E. x 400.
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detected in Gram-stained sections in any of the lesions. 2 days Lungs. Lesions were now more numerous than at 24 h and sufficiently large to be visible grossly on the surface and in the substance of the lungs as dark red blotches. For the first time there was necrosis of bronchial and bronchiolar epithelium and this destruction extended into the lamina propria (Fig. 2). Large areas of epithelium were also diffusely infiltrated by PMN, even in airways showing no other lesions. Locally the lumina of affected bronchi contained sloughed necrotic cellular debris and PMN. In a few regions the necrotizing process involving the airway wall coalesced with a lesion in surrounding alveoli. The alveolar changes remained as isolated nodules in which affected alveoli were distended by PMN and necrotic alveolar epithelial cells. Capillaries of the interalveolar septa were congested and alveoli adjacent to the nodules often contained oedema fluid. Between affected areas the lung tissue was structurally normal. Inflammatory changes in pulmonary arteries were first observed at this stage, and were a common and characteristic lesion. In many large and medium-sized branches of the pulmonary artery margination and adherence of leucocytes to the endothelium was taking place. Leucocytes also penetrated into a subendothelial position at some sites and this resulted in distortion and lifting of the endothelial cells from the underlying tissue (Fig. 3, 4). The majority of the invading cells were PMN but small lymphocytes were also present. Many lymphocytes remained in this location, but others together with large numbers of PMN migrated into the tunica media, where they forced apart the layers of smooth muscle cells, and also formed a heavy infiltration in the adventitial connective tissue of the artery. Pulmonary veins were normal at this stage. Nasal mucosa. There were a few areas of necrosis of the epithelium, and exudate
consisting of PMN and sloughed epithelial cells was present on the mucosal surface. The destructive changes also involved the more superficial tissue of the submucosa. Trachea.-In both rabbits there were isolated foci of necrosis and PMN infiltration in the tracheal epithelium. The lesions were similar to those in bronchi and also extended into the submucosa. The blood vessels of the submucosa in affected areas were greatly dilated and engorged with blood. Cervical and bronchial lymph nodes.A few lesions were found in these nodes, the bronchial being the more severely affected. They varied from tiny foci of necrosis of lymphoid tissue and PMN aggregation in the germinal centres of cortical follicles to coalescing areas of necrosis surrounded by PMN up to 1 mm in diameter extending into the paracortical regions and medullary cords. In the heavily infected bronchial nodes the medullary sinuses were distended by PMN, and these cells and lymphocytes also migrated into the loose connective tissue and fat surrounding the node capsule. The mesenteric lymph nodes were histologically normal at this stage. Spleen.-Necrotic foci with PMN, similar to those seen in the lymph nodes, were found in a few lymphoid nodules and occasionally these had spread to involve the adjacent red pulp. Liver.-Lesions were scattered throughout the liver. They took the form of nodules, usually about 005 mm in size, less commonly up to 015 mm, and had no regular lobular distribution. The centre of the nodule contained liver cells which had undergone coagulation necrosis and PMN, and macrophages and activated Kupffer cells were often present peripherally (Fig. 5). In the largest nodules there were also degenerating PMN. Hepatic tissue in the immediate vicinity of these nodules appeared normal. 3 days
Lungs-.By this stage the overall area of damaged lung tissue had increased
PATHOGENESIS AND PATHOLOGY OF RESPIRATORY TULARAEMIA
FIG. 5.-Characteristic lesion in liver at 2 days. H. and E. x 300. FIG. 6.-Lung 3 days after infection. Foci have increased in size, and in each area alveoli are distended by PMN. H. and E. x 40. FIG. 7.-Ventral nasal turbinate, 4 days. Necrosis has extended into the submucosa. There is destruction of portions of the bone, which are surrounded by inflammatory cells. H. and E. x 200.
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considerably from that at 2 days, but the lesions retained their patchy distribution (Fig. 6). Many bronchi and bronchioli had lost large areas of epithelium, their walls were completely necrotic and the lumina plugged by exudate and debris. These lesions frequently merged with similar necrotic areas of surrounding alveolar tissue. Thrombosis of peripheral lymphatics was also a common feature. Peripheral alveolar lesions had enlarged and now produced the pattern often seen in necrotizing bacterial pneumonia, of lobulated groups of necrotic alveolar tissue and PMN in which the ghost outlines of interalveolar septa could still be discerned. Alveoli immediately adjacent to these or to affected airways were filled with eosinophilic oedema fluid containing strands of fibrin. Pulmonary arteritis was prominent in all sections and small arteries were also affected. In addition to the changes noted at 2 days there was now disintegration of the endothelium and internal elastic lamina of the large branches at many of the sites of invasion by inflammatory cells. The connective tissue of the adventitia of these arteries was oedematous and contained fibrin. Occasionally penetration of PMN and lymphocytes beneath the endothelium was observed in a branch of the pulmonary vein, but this was a much less common finding than in arteries. Cervical, bronchial and mesenteric lymph nodes.-The cervical and bronchial nodes, which had probably become infected earlier than the mesenteric, were severely damaged, being almost totally necrotic and heavily infiltrated by PMN. In cons3quence very little of the follicular lymphoid tissue remained. Efferent lymphatics were occluded by thrombi, and even the surrounding fat was necrotic and contained numerous inflammatory cells. Changes in the mesenteric nodes were similar to those in the other nodes on the 2nd day and indicated that they were at an earlier stage of infection. Spleen.-The lesions were similar to
those found at 2 days, though larger and with more extension into the red pulp. Myocardium.-In one rabbit only there was myocarditis, with numerous PMN and lymphocytes between the muscle fibres in both ventricles. Muscle fibres were degenerating in the most severely affected areas. The endocardium was not involved. Liver.-Lesions were larger and more widespread than at 2 days, and typically had a central area of coagulation necrosis of hepatic cells together with PMN, surrounded by a zone 2-3 cells thick of lymphocytes and macrophages and enlarged Kupffer cells. Portal connective tissue was normal, except when directly involved by a peripheral granuloma. Nasal mucosa, soft palate, trachetz.-In one rabbit there was a large characteristic necrotic focus in the mucosa of the soft palate and in both animals there were a number of areas of necrosis in the nasal mucosa and trachea. 4 days
Lungs.-The acute necrotizing bronchitis and bronchiolitis were just as extensive as earlier, but at the periphery of many lesions there was now some fibroblastic activity. In small airways, organization of exudate in the lumen had resulted in bronchiolitis obliterans. As at 3 days, large thrombi were present in many peribronchial lymphatics. Alveolar lesions were larger than previously, some measuring 2 mm, and this appeared to be the result of breakdown of interalveolar septa. Damaged areas were surrounrlded by a narrow zone of lymphoreticular-cells and fibroblasts, a response probably developed to limit the outward spread of the acute destructive process. There were few intact PMN in the tissue at this stage, the majority having degenerated. Some interalveolar septa adjacent to the necrotic zones, not themselves affected, were thickened by proliferation of fibroblasts and accumulation of macrophages and lymphocytes. Pulmonary arteritis was still a charac-
PATHOGENESIS AND PATHOLOGY OF RESPIRATORY TULARAEMIA
teristic lesion, and in some branches, in addition to disruption of the internal elastic lamina, there were areas of medial necrosis. Phlebitis of occasional pulmonary vein branches was evident and appeared to be the result of direct extension from an adjacedit inflammatory focus. Leakage of proteinaceous fluid occurred from both arteries and veins into their adventitia and surrounding alveoli. Nasal mucosa and trachea.-Both rabbits had extensive lesions in these sites, which had destroyed not only epithelium but also much of the underlying tissue. In the nose there was destruction of portions of the turbinate bone (Fig. 7) and thrombi had formed in some branches of the venous plexus of the submucosa. Liver.-Nodules were structurally the same as at the earlier stages but they had enlarged to up to 03 mm in diameter. PMN and hepatic cells had degenerated completely, so that the centre of each nodule consisted of eosinophilic amorphous material. The peripheral cells were lymphocytes and activated Kupffer cells. Thrombi were present in a few branches of the portal vein. Kidneys. A small number of casts was present in distal tubules and collecting ducts but other renal tissue was morphologically normal. Spleen.-There was a great increase in the inflammatory reaction between the 3rd and 4th days, and the necrotic foci had formed large coalescing regions of tissue destruction which depleted the spleen of much of its lymphoid tissue. Cervical, bronchial and mesenteric lymph nodes.-Necrotizing and purulent lymphadenitis involved all the nodes on a similar scale to that seen at 3 days.
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tion of the infective particles, were the alveolar ducts and alveoli arising immediately from them. This is probably related to the small size of the particles generated by the Collison spray, 90%/ of which are 1-2 ,um in diameter (May, 1966). The initial distribution was thus similar to that in respiratory bronchioli and alveolar ducts after infection of Rhesus monkeys with F. tularensis in particles less than 5 /tm in size by White et al. (1962, 1964). The earliest change in the monkey lungs was respiratory bronchiolitis, the foci of alveolar necrosis not appearing until the 2nd day. This difference may be due to the slight anatomical differences between the 2 species, since the respiratory bronchioli of the rabbit are extremely short compared with those of the monkey. Limited immunofluorescence studies on the rabbit lungs showed groups of organisms at the alveolar duct level 1, 5 and 10 h after infection. It was not clear whether these organisms were being phagocytosed, though phagocytosis of F. tularensis in monkey lungs has been reported by White et al. (1964) as early as 20 min after exposure. Organisms with the typical morphology of F. tularensis were seen by electron microscopy within PMN and macrophages in alveoli of rabbits 24 h after exposure to an aerosol of the bacteria, though their ultimate fate in these cells could not be determined (Baskerville and Hambleton, unpublished observations). The virulent Schu-S4 strain of F. tularensis, in common with many other highly pathogenic bacteria, caused necrosis of bronchial, bronchiolar and alveolar epithelium. The typical areas of necrosis and infiltration by PMN in peripheral alveoli developed at least 24 h earlier than Control rabbits changes in the walls of airways. The Lesions were not detected in the organs mechanism by which the bacteria damage or lymph nodes of any of the 4 control epithelium is not known, but it may be by attachment to cell surfaces, by actual animals. penetration, or by the action of diffusible DISCUSSION toxic substances which they may produce. The earliest sites of reaction in the Lesions were present in the nasal mucosa lung, corresponding to the site of deposi- and trachea of the rabbits from the 2nd
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day onwards, but apparently were not found in the upper respiratory tract of monkeys infected by an aerosol of similar particle size (White et al., 1964). This may be due to the relative resistance of the monkey to tularaemia (White et al., 1962). In the rabbits the bacteria were removed from the nose, pharynx and trachea to the cervical lymph nodes where multiplication occurred and resulted in further dissemination of lesions. Once established, the disease in the rabbit became a typical necrotizing bronchopneumonia which spread rapidly to involve much of the lung by the 4th day. Its histological features were similar to those of pneumonia caused by bacteria of the genera Pasteurella, Salmonella and Staphylococcus in a variety of animals. Since rabbits rarely survive later than the 4th day after such infecting doses of bacteria the reparative tissue response was minimal and was represented at that stage by proliferation of fibroblasts, early organization of exudate, and the appearance of small numbers of lymphocytes at the periphery of lesions. The nodules in the liver and spleen were similar to those described previously in the rabbit and other animals after natural and experimental infection (Lillie and Francis, 1937a). Liver lesions did not generally appear until at least 24 h later than lung lesions, due presumably to the indirect route by which organisms reach the liver after respiratory infection. The liver may become infected either from the intestine after swallowing of bacteria or by blood-borne organisms derived ultimately from lymph nodes and lungs. The bronchial and cervical lymph nodes showed evidence of infection at 2 days, but there were no lesions in mesenteric nodes before the third day. It is not known whether F. tularensi.s can gain direct access to the blood through pulmonary capillaries. The presence of a few foci of PMN in the liver of one rabbit at the unusually early stage of 19 h was confirmed as a genuine response by the isolation of F. tularensis from adjacent
liver tissue. Since the liver changes are minor in comparison with the overwhelming lung damage, it is probable that they play little part in determining the outcome of the disease. The pulmonary arteritis, which was such a prominent feature of the lung changes, is recorded in the necropsy reports of some of the fatal human cases of tularaemia reviewed by Lillie and Francis (1 937b). It has not been mentioned in the generally brief earlier description of experimental tularaemia in animals, except in rabbits given two i.v. injections of irradiated, killed F. tularensis (Finegold et al., 1969). The pathogenesis of the lesions in the present study is obscure. They differed from those of Finegold et al. and from pulmonary arteritis induced in rabbits by meningococcal toxin (Brunson, Gamble and Thomas, 1955) in that there was no fibrinoid necrosis of the intima and wall and no thrombosis. The human infections in which arteritis was present were all of some weeks' duration and involved smaller arterial brainches than in the present rabbits. In the tularaemic rabbits there were no changes in arteries in organs other than the lung, or in renal glomeruli, which would be expected if the changes were mediated by immune complexes. The lesions were, however, identical to those described in the pulmonary arteries of rabbits with acute serum sickness induced by i.v. injections of large volumes of horse serum (Leber and McCluskey, 1974), though these took up to 10 days to develop, whereas they were present in the rabbits with tularaemia from the 2nd to the 4th day of infection. However, Wilens (1965) induced serum sickness-like lesions in the pulmonary arteries and to a lesser degree in the pulmonary veins of a small number of rabbits given homologous serum i.v., indicating that an immunological component may not be necessary for their development. His suggested explanation for the phenomenon was that the high blood pressure in the pulmonary arteries plays a part in the initiation of the
PATHOGENESIS AND PATHOLOGY OF RESPIRATORY TULARAEMIA
lesion. Acute changes in pulmonary arteries, though often accompanied by thrombosis, have also been described in fatal human septicaemia caused by bacteria such as Pseudomonas (Rabin et al., 1961). These findings and those in the present study indicate that pulmonary arteritis may be important, not only in tularaemia, but also in the pathogenesis of septicaemia and pneumonia due to other bacteria. REFERENCES BLACKFORD, S. D. & CASEY, C. J. (1941) Pleuropulmonary Tularaemia. Arch8. intern. Med., 67, 43. BRUNSON, J. G., GAMBLE, C. N. & THOMAS, L. (1955) Morphologic Changes in Rabbits Following the Intravenous Administration of Meningococcal Toxin. I. The Effects Produced in Young and in Mature Animals by a Single Injection. Am. J. Path., 31, 489. DowNs, C. M., CORIELL, L. L., PINCHOT, G. B., MAUMENEE, E., KLAUBER, A., CHAPMAN, S. S. & OWEN, B. (1947) Studies on Tularaemia. I. The Comparative Susceptibility of Various Laboratory Animals. J. Immunol., 56, 217. EIGELSBACH, H. T., BRAUM, W. & HERRING, R. D. (1951) Studies on the Variation of Bacterium tularense. J. Bact., 61, 557. FINEGOLD, M. J., PULLIAM, J. D., LANDAY, M. E. & WRIGHT, G. G. (1969) Pathological Changes in Rabbits Injected with P. tularen8i8 Killed by Ionizing Radiation. J. inf. Di8., 119, 635. HAMBLETON, P., EVANS, C. G. T., HOOD, A. M. & STRANGE, R. E. (1974) Vaccine Potencies of the Live Vaccine Strain of Francisella tularensis and Isolated Bacterial Components. Br. J. exp. Path., 55, 363. HENDERSON, D. W. (1952) An Apparatus for the Study of Airborne Infection. J. Hyg., Camb., 50, 53. JELLISON, W. L. & PARKER, R. R. (1945) Rodents, Rabbits and Tularaemia in North America: Some Zoological and Epidemiological Considerations. Am. J. trop. Med., 25, 349.
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LANDAY, M. E., WRIGHT, G. G., PULLIAM, J. D. & FINEGOLD, M. J. (1968) Toxicity of P. tularensis Killed by Ionizing Radiation. J. Bact., 96, 804. LEBER, P. D. & MCCLUSKEY, R. T. (1974) Immune Complex Diseases. In The Inflammatory Process, Vol. III, 2nd edition. Ed. B. W. Zweifach, L. Grant & R. T. McCluskey. New York: Academic Press. p. 401. LILLIE, R. D. & FRANCIS, E. (1937a) The Pathology of Tularaemia in the Black-tailed Jack Rabbit (Lepus sp.) In The Pathology of Tularaemia. National Institutes of Health Bulletin No. 167, p. 115. LILLIE, R. D. & FRANCIS, E. (1937b) The Pathology of Tularaemia in Man. In The Pathology of Tularaemia. National Institutes of Health Bulletin No. 167, p. 1. MAY, K. R. (1966) Multistage Liquid Impinger. Bact. Revs., 30, 559. MAY, K. R. & HARPER, G. J. (1957) The Efficiency of Various Liquid Impinger Samplers in Bacterial Aerosols. Br. J. indust. Med., 14, 287. MCCRUMB, F. R. (1961) Aerosol Infection of Man with Pasteurella tularensis. Bact. Revs., 25, 262. RABIN, E. R., GRABER, C. D., VOGEL, E. H., FINKELSTEIN, R. A. & TUMBUSCH, W. A. (1961) Fatal Pseudomonas Infection in Burned Patients. A Clinical, Bacteriologic and Anatomic Study. New Engl. J. Med., 265, 1225. SHARER, J. M., KLEIN, F. & LINCOLN, R. E. (1968) Growth and Metabolism of Live Vaccine Strain of Pasteurella tularensis. Appl. Microbiol., 16, 855. STRANGE, R. E., BENBOUGH, J. E., HAMBLETON, P. & MARTIN, K. L. (1974) Methods for the Assessment of Microbial Populations Recovered from Enclosed Aerosols. J. gen. Mierobiol., 72, 117. WHITE, J. D., McGAVRIN, M. H., PRICKETT, P. A., TULIS, J. J. & EIGELsACH, H. T. (1962) Morphologic and Immunohistochemical Studies of the Pathogenesis of Infection and Antibody Formation Subsequent to Vaccination of Macaca iru with an Attenuated Strain of Pasteurella tularensis. II. Aerogenic Vaccination. Am. J. Path., 41, 405. WHITE, J. D., ROONEY, J. R., PRICKETT, P. A., DERRENBACHER, E. B., BEARD, C. W. & GRIFFITH, W. R. (1964) Pathogenesis of Experimental Respiratory Tularaemia in Monkeys. J. inf. Dis., 114, 277. WILENS, S. L. (1965) Enhancement of Serum Sickness Lesions in Rabbits with Pressor Agents. Archs. Path., 80, 590.
PATHOGENESIS AND PATHOLOGY OF RESPIRATORY TULARAEMIA IN THE RABBIT A. BASKERVILLE AND P. HAMBLETON Frcomtt the Mlicrobiological Researcn Establishment, Porton, Salisbury, W'Viltshire Received for publication January 27, 1976
Summary.-The development of pathological lesions in the organs of rabbits was examined at intervals from 1 h to 4 days after aerosol infection with Francisella tularensis. The earliest change, accumulation of polymorphonuclear leucocytes (PMN) in pulmonary alveolar ducts, occurred at 19 h. From the 2nd day multiple foci of necrosis and PMN infiltration were present in large airways and alveoli throughout the lungs and progressively increased in size. Pulmonary arteritis was a prominent feature of the infection. Areas of necrosis were present in the nasal mucosa, pharynx and trachea, and pyogranolomatous lesions consistently developed in the liver, spleen, and lymph nodes.
Tularaemia, caused by the Gramnegative bacterium Francisella tularensis, is an important zoonosis in many parts of the world. The disease occurs in a large number of feral and domestic animals and one of the most important hosts which serves as a source of human infection is the rabbit (Jellison and Parker, 1945). Epizootics of tularaemia develop not infrequently in rabbits, hares and rodents in Europe, Russia and North America, and each year there are a number of fatal human cases associated with such outbreaks. Since the rabbit is a natural host for F. tularensis this species was used as the experimental animal in an investigation of the early clinico-pathological and histopathological changes which take place in tularaemia, and of the modification of the disease bv vaccination. Inhalation of F. tularensis in aerosols is thought to be one of the commonest modes of human infection, and this frequently results in pneumonia (McCrumb, 1961). To simulate this route of infection as closely as possible susceptible rabbits were exposed to aerosols of virulent F. tularensis to provide basic information for subsequent studies on animals which had been vaccinated
against tularaemia and challenged with virulent organisms. The pathology of fatal tularaemia in man and of the experimental disease in rodents and monkeys has been described by a number of workers since the disease was first recognized in the early years of this century (Lillie and Francis, 1937a, b; Blackford and Casey, 1941; Downs et al., 1947; White et al., 1964; Landay et al., 1968). Published information on the pathology of tularaemia in the rabbit is confined to the terminal condition following subcutaneous inoculation of the organism (Lillie and Francis, 1937a) and to the peracute disease induced by a vaccine strain of F. tularensis injected i.v. (Finegold et al., 1969). This communication describes the sequential histopathological changes occurring in the organs of rabbits infected by an aerosol containing the virulent Schu-S4 (Eigelsbach, Braum and Herring, 1951) strain of F. tularensis. MATERIALS AND METHODS
Micro-organism-Francisella tularensis, virulent Schu-S4 strain (Eigelsbach et al., 1951) was used. Growth of bacteria and preparation of washed
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suspensions.-Bacteria were grown in batch culture in the medium of Sharer, Klein and Lincoln (1968), harvested and washed twice with 1% (w/v) peptone + 0-1% (w/v) cysteine HC1 (cysteine broth) and resuspended in cysteine broth as described by Hambleton et al. (1974). For aerosolization bacteria were resuspended in cysteine broth containing antifoam agent (Strange et al., 1972). Determination of numbers of viable bacteria.Numbers of viable F. tularen8si in suspensions of bacteria or tissue homogenates were determined as described by Strange et al. (1972). Animals.-Seventeen New Zealand White male and female rabbits were used. Infection of animals with F. tularensis.Aerosols of F. tularensis Schu-S4 generated with a 3-jet Collision spray from suspensions containing 5 x 109 or 2 x 1011 viable bacteria/ml were held in a Henderson (1952) apparatus and sampled for 1 min with a Porton Raised Impinger (May and Harper, 1957) having a flow rate of 11 I/min and containing 10 ml of cysteine broth + antifoam agent. Thirteen rabbits were infected by exposure for 1 min to bacterial aerosols containing 2 x 105 or 4 x 108 viable bacteria/l. The calculated inhaled retention doses of viable bacteria given to 10 rabbits were 1-2 x 105andtothe3others2-4 x 108. Four rabbits served as uninfected controls. Necropsy stages and procedure.-Preliminary studies showed that in the rabbit the disease runs an acute course and usually proves fatal after 4 or 5 days. Rabbits were therefore killed during the first 4 days after infection. The 3 given a dose of 2-4 x 108 organisms were killed, 1, 5 and 10 h after exposure, and of the animals given 1-2 x 105 bacteria 2 were sacrificed at each of the following stages: 19 h, 24 h, 2, 3 and 4 days. One control rabbit was killed each day, from the first to the 4th day. All animals were killed by i.v. or i.p. injection of pentobarbitone sodium. At necropsy carried out immediately after death the following organs were removed and portions fixed in 10% formol saline: ventral nasal turbinate bones, soft palate and palatine tonsils, larynx, trachea, lungs, heart, liver, spleen, kidneys, small intestine and brain. The cervical, bronchial and mesenteric lymph nodes were fixed for 1 h in Carnoy's fluid and subsequently transferred to 80% alcohol. The larynx and turbinate bones were decalcified in modified Gooding and Stewart's fluid. All tissues were processed by standard methods and embedded in paraffin wax. Sections cut at 5 ,um were stained with haematoxylin and eosin. Selected sections were stained with Mallory's phosphotungstic acid haematoxylin, by Gordon and Sweet's method for reticulin, with methyl green-pyronin, by periodic acid-Schiff, by Gram's stain, and by the combined Verhoeff's elastin and Van Gieson methods.
Bacteriological examination of homogenized tis8Ues.-Portions of organs excised at necropsy from infected rabbits were homogenized in a small volume (about 3 ml) of cysteine broth and viable F. tularensis detected as described above. Bacterial isolates were confirmed as F. tularensis on the basis of colony morphology, Gram staining and agglutination by rabbit anti-F. tularensis serum. RESULTS
Clinically the animals were dull and had slightly elevated temperatures by the 2nd day. On the 3rd day food intake was greatly reduced and there was diarrhoea and pyrexia in excess of 40.50. Pathological changes were not detected in the lungs or other organs of the rabbits killed at 1, 5 and 10 h after infection. For the other animals, changes were not found in organs not specified in the following description. 19 h
Lungs.-The earliest recognizable lesion developed at this stage and consisted of a small number of peripherally situated foci of polymorphonuclear leucocytes (PMN), principally in the region of alveolar ducts or affecting a group of several alveoli. F. tularensis organisms could not be found in these foci or at other sites in Gram-stained sections, probably because of their extremely small size and similar staining properties to those of the tissue, though the organism was isolated consistently from the lungs at all stages of infection from 19 h onwards. Liver.-In one animal only, the liver contained a few small accumulations of PMN. These had no consistent lobular distribution and there was no evidence of damage to hepatic cells. 24 h
Lungs.-Numerous small foci of PMN were scattered peripherally and involved alveolar ducts and alveoli (Fig. 1). Alveolar capillaries in affected regions were intensely congested. In a number of large bronchi and bronchioli there was localized infiltration of the epithelium and lamina propria by PMN. Bacteria could not be
PATHOGENESIS AND PATHOLOGY OF RESPIRATORY TULARAEMIA
341
FIG. 1. Typical focus of PMN infiltration and necrosis in lung at 24 h. H. and E. x 120. FIG. 2.-2 days after infection. Focal necrosis of bronchial epithelium extends into the surrounding tissue. H. and E. x 50. FIG. 3.-Pulmonary arteritis on 2nd day. There is cellular infiltration accompanied by gross distortion of the endothelial cell layer. H. and E. x 110. FIG. 4.-Higher magnification of Fig. 3. Inflammatory cells have accumulated beneath the endothelium, which is disrupted at several points, and are invading the media. H. and E. x 400.
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A. BASKERVILLE AND P. HAMBLETON
detected in Gram-stained sections in any of the lesions. 2 days Lungs. Lesions were now more numerous than at 24 h and sufficiently large to be visible grossly on the surface and in the substance of the lungs as dark red blotches. For the first time there was necrosis of bronchial and bronchiolar epithelium and this destruction extended into the lamina propria (Fig. 2). Large areas of epithelium were also diffusely infiltrated by PMN, even in airways showing no other lesions. Locally the lumina of affected bronchi contained sloughed necrotic cellular debris and PMN. In a few regions the necrotizing process involving the airway wall coalesced with a lesion in surrounding alveoli. The alveolar changes remained as isolated nodules in which affected alveoli were distended by PMN and necrotic alveolar epithelial cells. Capillaries of the interalveolar septa were congested and alveoli adjacent to the nodules often contained oedema fluid. Between affected areas the lung tissue was structurally normal. Inflammatory changes in pulmonary arteries were first observed at this stage, and were a common and characteristic lesion. In many large and medium-sized branches of the pulmonary artery margination and adherence of leucocytes to the endothelium was taking place. Leucocytes also penetrated into a subendothelial position at some sites and this resulted in distortion and lifting of the endothelial cells from the underlying tissue (Fig. 3, 4). The majority of the invading cells were PMN but small lymphocytes were also present. Many lymphocytes remained in this location, but others together with large numbers of PMN migrated into the tunica media, where they forced apart the layers of smooth muscle cells, and also formed a heavy infiltration in the adventitial connective tissue of the artery. Pulmonary veins were normal at this stage. Nasal mucosa. There were a few areas of necrosis of the epithelium, and exudate
consisting of PMN and sloughed epithelial cells was present on the mucosal surface. The destructive changes also involved the more superficial tissue of the submucosa. Trachea.-In both rabbits there were isolated foci of necrosis and PMN infiltration in the tracheal epithelium. The lesions were similar to those in bronchi and also extended into the submucosa. The blood vessels of the submucosa in affected areas were greatly dilated and engorged with blood. Cervical and bronchial lymph nodes.A few lesions were found in these nodes, the bronchial being the more severely affected. They varied from tiny foci of necrosis of lymphoid tissue and PMN aggregation in the germinal centres of cortical follicles to coalescing areas of necrosis surrounded by PMN up to 1 mm in diameter extending into the paracortical regions and medullary cords. In the heavily infected bronchial nodes the medullary sinuses were distended by PMN, and these cells and lymphocytes also migrated into the loose connective tissue and fat surrounding the node capsule. The mesenteric lymph nodes were histologically normal at this stage. Spleen.-Necrotic foci with PMN, similar to those seen in the lymph nodes, were found in a few lymphoid nodules and occasionally these had spread to involve the adjacent red pulp. Liver.-Lesions were scattered throughout the liver. They took the form of nodules, usually about 005 mm in size, less commonly up to 015 mm, and had no regular lobular distribution. The centre of the nodule contained liver cells which had undergone coagulation necrosis and PMN, and macrophages and activated Kupffer cells were often present peripherally (Fig. 5). In the largest nodules there were also degenerating PMN. Hepatic tissue in the immediate vicinity of these nodules appeared normal. 3 days
Lungs-.By this stage the overall area of damaged lung tissue had increased
PATHOGENESIS AND PATHOLOGY OF RESPIRATORY TULARAEMIA
FIG. 5.-Characteristic lesion in liver at 2 days. H. and E. x 300. FIG. 6.-Lung 3 days after infection. Foci have increased in size, and in each area alveoli are distended by PMN. H. and E. x 40. FIG. 7.-Ventral nasal turbinate, 4 days. Necrosis has extended into the submucosa. There is destruction of portions of the bone, which are surrounded by inflammatory cells. H. and E. x 200.
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A. BASKERVILLE AND P. HAMBLETON
considerably from that at 2 days, but the lesions retained their patchy distribution (Fig. 6). Many bronchi and bronchioli had lost large areas of epithelium, their walls were completely necrotic and the lumina plugged by exudate and debris. These lesions frequently merged with similar necrotic areas of surrounding alveolar tissue. Thrombosis of peripheral lymphatics was also a common feature. Peripheral alveolar lesions had enlarged and now produced the pattern often seen in necrotizing bacterial pneumonia, of lobulated groups of necrotic alveolar tissue and PMN in which the ghost outlines of interalveolar septa could still be discerned. Alveoli immediately adjacent to these or to affected airways were filled with eosinophilic oedema fluid containing strands of fibrin. Pulmonary arteritis was prominent in all sections and small arteries were also affected. In addition to the changes noted at 2 days there was now disintegration of the endothelium and internal elastic lamina of the large branches at many of the sites of invasion by inflammatory cells. The connective tissue of the adventitia of these arteries was oedematous and contained fibrin. Occasionally penetration of PMN and lymphocytes beneath the endothelium was observed in a branch of the pulmonary vein, but this was a much less common finding than in arteries. Cervical, bronchial and mesenteric lymph nodes.-The cervical and bronchial nodes, which had probably become infected earlier than the mesenteric, were severely damaged, being almost totally necrotic and heavily infiltrated by PMN. In cons3quence very little of the follicular lymphoid tissue remained. Efferent lymphatics were occluded by thrombi, and even the surrounding fat was necrotic and contained numerous inflammatory cells. Changes in the mesenteric nodes were similar to those in the other nodes on the 2nd day and indicated that they were at an earlier stage of infection. Spleen.-The lesions were similar to
those found at 2 days, though larger and with more extension into the red pulp. Myocardium.-In one rabbit only there was myocarditis, with numerous PMN and lymphocytes between the muscle fibres in both ventricles. Muscle fibres were degenerating in the most severely affected areas. The endocardium was not involved. Liver.-Lesions were larger and more widespread than at 2 days, and typically had a central area of coagulation necrosis of hepatic cells together with PMN, surrounded by a zone 2-3 cells thick of lymphocytes and macrophages and enlarged Kupffer cells. Portal connective tissue was normal, except when directly involved by a peripheral granuloma. Nasal mucosa, soft palate, trachetz.-In one rabbit there was a large characteristic necrotic focus in the mucosa of the soft palate and in both animals there were a number of areas of necrosis in the nasal mucosa and trachea. 4 days
Lungs.-The acute necrotizing bronchitis and bronchiolitis were just as extensive as earlier, but at the periphery of many lesions there was now some fibroblastic activity. In small airways, organization of exudate in the lumen had resulted in bronchiolitis obliterans. As at 3 days, large thrombi were present in many peribronchial lymphatics. Alveolar lesions were larger than previously, some measuring 2 mm, and this appeared to be the result of breakdown of interalveolar septa. Damaged areas were surrounrlded by a narrow zone of lymphoreticular-cells and fibroblasts, a response probably developed to limit the outward spread of the acute destructive process. There were few intact PMN in the tissue at this stage, the majority having degenerated. Some interalveolar septa adjacent to the necrotic zones, not themselves affected, were thickened by proliferation of fibroblasts and accumulation of macrophages and lymphocytes. Pulmonary arteritis was still a charac-
PATHOGENESIS AND PATHOLOGY OF RESPIRATORY TULARAEMIA
teristic lesion, and in some branches, in addition to disruption of the internal elastic lamina, there were areas of medial necrosis. Phlebitis of occasional pulmonary vein branches was evident and appeared to be the result of direct extension from an adjacedit inflammatory focus. Leakage of proteinaceous fluid occurred from both arteries and veins into their adventitia and surrounding alveoli. Nasal mucosa and trachea.-Both rabbits had extensive lesions in these sites, which had destroyed not only epithelium but also much of the underlying tissue. In the nose there was destruction of portions of the turbinate bone (Fig. 7) and thrombi had formed in some branches of the venous plexus of the submucosa. Liver.-Nodules were structurally the same as at the earlier stages but they had enlarged to up to 03 mm in diameter. PMN and hepatic cells had degenerated completely, so that the centre of each nodule consisted of eosinophilic amorphous material. The peripheral cells were lymphocytes and activated Kupffer cells. Thrombi were present in a few branches of the portal vein. Kidneys. A small number of casts was present in distal tubules and collecting ducts but other renal tissue was morphologically normal. Spleen.-There was a great increase in the inflammatory reaction between the 3rd and 4th days, and the necrotic foci had formed large coalescing regions of tissue destruction which depleted the spleen of much of its lymphoid tissue. Cervical, bronchial and mesenteric lymph nodes.-Necrotizing and purulent lymphadenitis involved all the nodes on a similar scale to that seen at 3 days.
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tion of the infective particles, were the alveolar ducts and alveoli arising immediately from them. This is probably related to the small size of the particles generated by the Collison spray, 90%/ of which are 1-2 ,um in diameter (May, 1966). The initial distribution was thus similar to that in respiratory bronchioli and alveolar ducts after infection of Rhesus monkeys with F. tularensis in particles less than 5 /tm in size by White et al. (1962, 1964). The earliest change in the monkey lungs was respiratory bronchiolitis, the foci of alveolar necrosis not appearing until the 2nd day. This difference may be due to the slight anatomical differences between the 2 species, since the respiratory bronchioli of the rabbit are extremely short compared with those of the monkey. Limited immunofluorescence studies on the rabbit lungs showed groups of organisms at the alveolar duct level 1, 5 and 10 h after infection. It was not clear whether these organisms were being phagocytosed, though phagocytosis of F. tularensis in monkey lungs has been reported by White et al. (1964) as early as 20 min after exposure. Organisms with the typical morphology of F. tularensis were seen by electron microscopy within PMN and macrophages in alveoli of rabbits 24 h after exposure to an aerosol of the bacteria, though their ultimate fate in these cells could not be determined (Baskerville and Hambleton, unpublished observations). The virulent Schu-S4 strain of F. tularensis, in common with many other highly pathogenic bacteria, caused necrosis of bronchial, bronchiolar and alveolar epithelium. The typical areas of necrosis and infiltration by PMN in peripheral alveoli developed at least 24 h earlier than Control rabbits changes in the walls of airways. The Lesions were not detected in the organs mechanism by which the bacteria damage or lymph nodes of any of the 4 control epithelium is not known, but it may be by attachment to cell surfaces, by actual animals. penetration, or by the action of diffusible DISCUSSION toxic substances which they may produce. The earliest sites of reaction in the Lesions were present in the nasal mucosa lung, corresponding to the site of deposi- and trachea of the rabbits from the 2nd
346
A. BASKERVILLE AND P. HAMBLETON
day onwards, but apparently were not found in the upper respiratory tract of monkeys infected by an aerosol of similar particle size (White et al., 1964). This may be due to the relative resistance of the monkey to tularaemia (White et al., 1962). In the rabbits the bacteria were removed from the nose, pharynx and trachea to the cervical lymph nodes where multiplication occurred and resulted in further dissemination of lesions. Once established, the disease in the rabbit became a typical necrotizing bronchopneumonia which spread rapidly to involve much of the lung by the 4th day. Its histological features were similar to those of pneumonia caused by bacteria of the genera Pasteurella, Salmonella and Staphylococcus in a variety of animals. Since rabbits rarely survive later than the 4th day after such infecting doses of bacteria the reparative tissue response was minimal and was represented at that stage by proliferation of fibroblasts, early organization of exudate, and the appearance of small numbers of lymphocytes at the periphery of lesions. The nodules in the liver and spleen were similar to those described previously in the rabbit and other animals after natural and experimental infection (Lillie and Francis, 1937a). Liver lesions did not generally appear until at least 24 h later than lung lesions, due presumably to the indirect route by which organisms reach the liver after respiratory infection. The liver may become infected either from the intestine after swallowing of bacteria or by blood-borne organisms derived ultimately from lymph nodes and lungs. The bronchial and cervical lymph nodes showed evidence of infection at 2 days, but there were no lesions in mesenteric nodes before the third day. It is not known whether F. tularensi.s can gain direct access to the blood through pulmonary capillaries. The presence of a few foci of PMN in the liver of one rabbit at the unusually early stage of 19 h was confirmed as a genuine response by the isolation of F. tularensis from adjacent
liver tissue. Since the liver changes are minor in comparison with the overwhelming lung damage, it is probable that they play little part in determining the outcome of the disease. The pulmonary arteritis, which was such a prominent feature of the lung changes, is recorded in the necropsy reports of some of the fatal human cases of tularaemia reviewed by Lillie and Francis (1 937b). It has not been mentioned in the generally brief earlier description of experimental tularaemia in animals, except in rabbits given two i.v. injections of irradiated, killed F. tularensis (Finegold et al., 1969). The pathogenesis of the lesions in the present study is obscure. They differed from those of Finegold et al. and from pulmonary arteritis induced in rabbits by meningococcal toxin (Brunson, Gamble and Thomas, 1955) in that there was no fibrinoid necrosis of the intima and wall and no thrombosis. The human infections in which arteritis was present were all of some weeks' duration and involved smaller arterial brainches than in the present rabbits. In the tularaemic rabbits there were no changes in arteries in organs other than the lung, or in renal glomeruli, which would be expected if the changes were mediated by immune complexes. The lesions were, however, identical to those described in the pulmonary arteries of rabbits with acute serum sickness induced by i.v. injections of large volumes of horse serum (Leber and McCluskey, 1974), though these took up to 10 days to develop, whereas they were present in the rabbits with tularaemia from the 2nd to the 4th day of infection. However, Wilens (1965) induced serum sickness-like lesions in the pulmonary arteries and to a lesser degree in the pulmonary veins of a small number of rabbits given homologous serum i.v., indicating that an immunological component may not be necessary for their development. His suggested explanation for the phenomenon was that the high blood pressure in the pulmonary arteries plays a part in the initiation of the
PATHOGENESIS AND PATHOLOGY OF RESPIRATORY TULARAEMIA
lesion. Acute changes in pulmonary arteries, though often accompanied by thrombosis, have also been described in fatal human septicaemia caused by bacteria such as Pseudomonas (Rabin et al., 1961). These findings and those in the present study indicate that pulmonary arteritis may be important, not only in tularaemia, but also in the pathogenesis of septicaemia and pneumonia due to other bacteria. REFERENCES BLACKFORD, S. D. & CASEY, C. J. (1941) Pleuropulmonary Tularaemia. Arch8. intern. Med., 67, 43. BRUNSON, J. G., GAMBLE, C. N. & THOMAS, L. (1955) Morphologic Changes in Rabbits Following the Intravenous Administration of Meningococcal Toxin. I. The Effects Produced in Young and in Mature Animals by a Single Injection. Am. J. Path., 31, 489. DowNs, C. M., CORIELL, L. L., PINCHOT, G. B., MAUMENEE, E., KLAUBER, A., CHAPMAN, S. S. & OWEN, B. (1947) Studies on Tularaemia. I. The Comparative Susceptibility of Various Laboratory Animals. J. Immunol., 56, 217. EIGELSBACH, H. T., BRAUM, W. & HERRING, R. D. (1951) Studies on the Variation of Bacterium tularense. J. Bact., 61, 557. FINEGOLD, M. J., PULLIAM, J. D., LANDAY, M. E. & WRIGHT, G. G. (1969) Pathological Changes in Rabbits Injected with P. tularen8i8 Killed by Ionizing Radiation. J. inf. Di8., 119, 635. HAMBLETON, P., EVANS, C. G. T., HOOD, A. M. & STRANGE, R. E. (1974) Vaccine Potencies of the Live Vaccine Strain of Francisella tularensis and Isolated Bacterial Components. Br. J. exp. Path., 55, 363. HENDERSON, D. W. (1952) An Apparatus for the Study of Airborne Infection. J. Hyg., Camb., 50, 53. JELLISON, W. L. & PARKER, R. R. (1945) Rodents, Rabbits and Tularaemia in North America: Some Zoological and Epidemiological Considerations. Am. J. trop. Med., 25, 349.
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LANDAY, M. E., WRIGHT, G. G., PULLIAM, J. D. & FINEGOLD, M. J. (1968) Toxicity of P. tularensis Killed by Ionizing Radiation. J. Bact., 96, 804. LEBER, P. D. & MCCLUSKEY, R. T. (1974) Immune Complex Diseases. In The Inflammatory Process, Vol. III, 2nd edition. Ed. B. W. Zweifach, L. Grant & R. T. McCluskey. New York: Academic Press. p. 401. LILLIE, R. D. & FRANCIS, E. (1937a) The Pathology of Tularaemia in the Black-tailed Jack Rabbit (Lepus sp.) In The Pathology of Tularaemia. National Institutes of Health Bulletin No. 167, p. 115. LILLIE, R. D. & FRANCIS, E. (1937b) The Pathology of Tularaemia in Man. In The Pathology of Tularaemia. National Institutes of Health Bulletin No. 167, p. 1. MAY, K. R. (1966) Multistage Liquid Impinger. Bact. Revs., 30, 559. MAY, K. R. & HARPER, G. J. (1957) The Efficiency of Various Liquid Impinger Samplers in Bacterial Aerosols. Br. J. indust. Med., 14, 287. MCCRUMB, F. R. (1961) Aerosol Infection of Man with Pasteurella tularensis. Bact. Revs., 25, 262. RABIN, E. R., GRABER, C. D., VOGEL, E. H., FINKELSTEIN, R. A. & TUMBUSCH, W. A. (1961) Fatal Pseudomonas Infection in Burned Patients. A Clinical, Bacteriologic and Anatomic Study. New Engl. J. Med., 265, 1225. SHARER, J. M., KLEIN, F. & LINCOLN, R. E. (1968) Growth and Metabolism of Live Vaccine Strain of Pasteurella tularensis. Appl. Microbiol., 16, 855. STRANGE, R. E., BENBOUGH, J. E., HAMBLETON, P. & MARTIN, K. L. (1974) Methods for the Assessment of Microbial Populations Recovered from Enclosed Aerosols. J. gen. Mierobiol., 72, 117. WHITE, J. D., McGAVRIN, M. H., PRICKETT, P. A., TULIS, J. J. & EIGELsACH, H. T. (1962) Morphologic and Immunohistochemical Studies of the Pathogenesis of Infection and Antibody Formation Subsequent to Vaccination of Macaca iru with an Attenuated Strain of Pasteurella tularensis. II. Aerogenic Vaccination. Am. J. Path., 41, 405. WHITE, J. D., ROONEY, J. R., PRICKETT, P. A., DERRENBACHER, E. B., BEARD, C. W. & GRIFFITH, W. R. (1964) Pathogenesis of Experimental Respiratory Tularaemia in Monkeys. J. inf. Dis., 114, 277. WILENS, S. L. (1965) Enhancement of Serum Sickness Lesions in Rabbits with Pressor Agents. Archs. Path., 80, 590.
