Protection Against Experimental Bordetella bronchiseptica

Transcription

1 INFECTION AND IMMUNITY, Aug. 1983, p /83/ $02.00/0 Copyright C 1983, Amerian Soiety for Mirobiology Vol. 41, No. 2 Protetion Against Experimental Bordetella bronhiseptia Infetion in Mie by Ative Immunization with Killed Vaine KACHIKO SEKIYA,'* MINORU KAWAHIRA,2t AND YASUKIYO NAKASE1 Department of Mirobiology, Shool of Pharmaeutial Sienes, Kitasato University, Minato-ku,1 and Department ofbateriology, Kitasato Institute,2 Tokyo 108, Japan Reeived 22 February 1983/Aepted 18 May 1983 Mie immunized with a killed vaine of phase I Brodetella bronhiseptia were hallenged with various numbers of virulent B. bronhiseptia by intraperitoneal, intraerebral, or intranasal routes. The ourse of infetion was ompared among these routes, and the protetive effet of vaination was quantitatively analyzed. In ddn mie infeted intraperitoneally with 1.8 x 108 ells (a. 80 times the 50% lethal dose [LD50]) the organisms rapidly inreased in the intraperitoneal fluid, spleen, and liver within few days and aused spleni atrophy, septiemia, and death. However, immunizations with 5 x 109 ells gave the mie a high agglutinin titer and suppressed the inrease in the number of organisms. With four immunizations, the lungs and livers were lear within 3 days, and with one or two immunizations, they were lear within 7 days. These immunizations effetively proteted the mie from death but did not protet them from spleni atrophy. In the intraerebral infetion with 1.4 x 106 ells (a. 1.2 x 105 LD50), the number of organisms rapidly inreased in the brain and aused enephalitis, spleni atrophy, and death. However, four or five immunizations ompletely suppressed the inrease in the brain and proteted the mie from death and spleni atrophy. After intranasal infetion with 4 x 106 ells (a. 25 LD50), the organisms rapidly inreased in the nasal avity and lungs and aused pneumonia and death. Immunization with 5 x 109 ells was effetive in learing the organisms from the lungs and in proteting against death and spleni atrophy. However, the organisms were not leared from the nasal avity for 60 to 150 days after the hallenge with as little as 102 ells, even in the mie with an agglutinin titer as high as 1:10,000. Several studies (3, 5, 6, 10-12, 18-20) have indiated that ertain strains of Bordetella bronhiseptia gave rise to a fatal infetion in laboratory animals after experimental inoulation and that immunization with a killed B. bronhiseptia vaine gives a signifiant degree of protetion against the lethal effets. Tanaka (19, 20) found that in mie the vaine was protetive against infetion by intraperitoneal (i.p.), subutaneous, and intravenous routes but it was poorly protetive against infetion by the intranasal (i.n.) route. Kendrik et al. (7) and Nakase (13) reported that intraerebral (i..) or i.p. infetions of mie were attenuated by immunization. In guinea pigs (3, 6, 10, 11), immunization with a killed B. bronhiseptia vaine was effetive in proteting against fatal infetions by the i.n. and i.p. routes. In the present paper, an attempt was made to ompare the ourse of infetion among various t Present address: Izumo Liverstok Hygiene Servie Center, Izumo, Shimane 693, Japan. routes, to analyze quantitatively the protetive effet of a killed B. bronhiseptia vaine, to larify the mehanisms of B. bronhiseptia infetion and immunity, and hene to develop a suitable mouse model. MATERIALS AND METHODS Mie. Speifi pathogen-free female 4-week-old ddn mie from a olony free of B. bronhiseptia raised at the Kitasato Institute were used. They were housed five in a age. Bateria. Phase I B. bronhiseptia strains L3, isolated from a piglet (14), and H-16, reeived from M. Ogata (Tokyo University), were used. These were passed several times through lungs or brains of mie. Immunization. To prepare a killed B. bronhiseptia vaine, we suspended ells ultured on haroal agar medium (1, 15) for 24 h in phosphate-buffered saline and inativated them with 0.1% Formalin. The vaine was adjusted to a density of 1010 ells per ml by eletrophotometer, Merthiolate (0.01%) was added, and the vaine was suitably diluted with phosphatebuffered saline before use. Mie reeived 0.5 ml by the i.p. route at eah injetion. 598

2 VOL. 41, 1983 a -4 4 a > O _4 v 5 _0 > _ O.-I o) 4 O 4) Xi ff 2 FIG. spleens with 5 and of Infe um for (ph 7.0 sion wa EXPERIMENTAL INFECTION WITH B. BRONCHISEPTICA 599 mie woere infeted by the i.p., i.., or i.n. route with the surviving mie whih were killed on day , 0.a )25, or ml, respetively, of the suitable posthallenge, the spleen weight was dereased dilutioni. For the i.n. inoulation, mie were lightly to one-half to one-third that of normal mie. In anesthe-tized with ether. Viabile ount. The suspension was tested for viable the mie immunized with 40 x 106 to 200 x 106 ells as soon as the inoulation was finished. The ells, a few organisms (a. 102) were deteted in infete I organs, i.e., spleen, lungs, liver, brain, and the spleen and liver. But none were deteted in nasal ; avity, were homogenized individually in a mor- the organs of mie immunized with 1,000 x 106 tar witl h 2 to 5 ml of Casamino Aids and a small to 5,000 x 106 ells. amountt of glass beads (0.17 to 0.18 mm in diameter) Effet of vaination on i.. infetion. In mie and asssayed for viable ount. The nasal avity was infeted by the i.. route with 1.4 x 106 ells (a. obtained by taking the whole upper jaw with the 1.2 x 106 LD50), the number of organisms in the maxilla by first stripping off the skin and then setion- *. ' 5 ing rotss-wise under the eyes. The organ emulsions brain rapidly inreased from ells 2 h after were de mimally diluted with Casamino Aids and infetion (day 0) to 107 on day 2. Immunization ml fron eah of three suitable dilutions was dropped with one to two doses of 5 x 109 ells gave no onto tv blood a 370C f ounte( four to were st Aggli tions of ells p Formal mixed reorded after standing the mixtures at 2 to 40C overnight. RESULTS l, Effet of vaination on i.p. infetion. When g unimmunized mie were infeted with 1.8 x 108 ells (a. 80 times the 50% lethal dose [LD50]) by the i.p. route, the number of organisms in the livers and spleens inreased rapidly and reahed 108 to 109 ells within 1 to 2 days. In the groups 2L\ immunized with one to two doses of 5 x 109 ells, the number of organisms in the livers and \( 1) 1) spleens gradually dereased and none were de- (2 teted on day 10 posthallenge. With four to five doses, the number of organisms rapidly de- (4 0 reased after hallenge and ould not be detet ed on days 2 to 3 (Fig. 1). Agglutinin titers at the time of hallenge were less than 1:10 in the Days after hallenge unimmunized group, whereas titers of 1:640, 1. Viable ounts of B. bronhiseptia in 1:1,280 to 1:2,560, 1:10,240, or 1:10,240 to and livers of mie immunized by the i.p. route 1:20,480 were found in animals reeiving one, x 109 ells in various doses at 1-week intervals * ' * * * unimmunized ontrol mie. Mie were hal- two, four, or five immunizatlons, respetively. lengedi14 days after the last immunization with 1.8 x When mie were hallenged with a sublethal 108 ellis of B. bronhiseptia L3. Throughout these dose (2.6 x 105 ells, a LD50), a few figures, eah point represents the average ount in organisms still persisted in the liver and spleen four to five mie. Symbols: *-*, livers of unim- even on day 20 in the unimmunized mie, but in munize d ontrol mie; *---@, spleens of unimmu- the mie immunized with 5 x 109 ells, none nized -ontrol mie; O-O, livers of immunized ould be deteted on day 1. mie; C)---O, spleens of immunized mie. Numbers in Table 1 shows the effets of immunization parenthkeses indiate the number of times mie were against i.p. hallenge. Most of the mie immu- with 5 x 109 ells. nized with over 200 x 106 ells survived hal- immunized lenge with 3.2 x 107 ells (a. 10 LD50). In the tion. Cultures grown on Bordet-Gengou medi- mie that died on day 3 or 4 posthallenge, the 20 h were suspended in 1% Casamino Aids spleen weight was less than half that of normal ) at a density of 1010 ells per ml. The suspen- mie (103.6 ± 9.8 mg). In the livers, spleens, and is deimally diluted with Casamino Aids, and heart blood, 108 to 109 ells were deteted. In vo haroal agar plates ontaining 5% horse protetion, as all the mie died on days 2 to 4 Lnd spread by Conradi pole. After inubation at posthallenge. However, immunization with )r 2 or 3 days, the number of olonies was four to five doses, whih gave high serum agglud. An average value was obtained by examining tinin titers (1:10, 240 to 1:20,480) at the time of five mie per group. The viable ounts on day 0 hallenge, proteted the mie from death. In this tarted 2 h after infetion. ' stination test. A0.5-ml portion of twofold dilu- ase, the organisms in the brain gradually inrserum and an equal volume of the antigen (0lo1 reased to about 1tO ells on days 2 to 3 poster ml), whih had been inativated with 0.1% hallenge and then dereased and disappeared [in in a density of 50 x 1010 ells per ml, were on days 7 to 14 (Fig. 2). and inubated for 2 h at 36 C. Results were Table 2 shows the effets of immunization

3 600 SEKIYA, KAWAHIRA, AND NAKASE TABLE 1. Survival rate, body and spleen weights, and the ounts of viable B. bronhiseptia in spleen, liver, and heart blood after i.p. hallenge in mie with various levels of immunization Immunization Challenge No. of viable organisms per organ dose dose Survived/ Day of death Body Spleen in.: (x196ells) (viable ells)' (day kilied) wt (g) wt (mg) Spleen Liver blood 5, x /10 (20) NDd ND ND 1, x /10 (20) ND ND ND x 107 9/10 (20) x x 102 ND x 107 3/10 (20) x x 102 ND x 107 1/ x x x x 107 0/ x x x x 106 3/5 (20) x x 102 ND x 105 5/5 (20) x x 102 ND a Mie were hallenged by the i.p. route with B. bronhiseptia L3 14 days after immunization. "At 14 days after hallenge. Average of five mie (all the surviving mie in the ase of less than five mie per group). d ND, Not deteted in ml of suspension (<100 ells per organ). against i.. infetion with 5.6 x 104 ells (a. 2,000 LD50). Immunization with 1,000 x 106 ells gave partial protetion. A remarkable redution in spleen weight to about one-third that of normal mie was noted in those mie whih died on day 4 posthallenge. The number of organisms in the brain was 106 to 107 ells, but none were deteted in the spleen or heart blood. In the surviving mie, the spleens were almost normal size on day 20 postinfetion, and no organisms were deteted in the brain. Effet of vaination on i.n. infetion. Mie infeted by the i.n. route with 6.8 x 107 ells died within 7 days, but prior immunization with over 200 x 106 ells ompletely abolished the lethal effets. The spleen weights of mie whih died on days 2 to 4 posthallenge were about one-half to one-third that of normal mie. The numbers of organisms found in the nasal avity and in the lungs were 106 and 109, respetively, but 105 ells were found in the spleen and in heart blood, suggesting that the mie died by pneumonia rather than septiemia. The numbers of organisms in the nasal avities of mie killed on day 20 posthallenge were about 1/10 the number found in mie that died on days 2 to 4 posthallenge. The number of organisms in the lungs of killed mie was markedly less than that found in dead mie, and it was observed that the number of residual ells dereased as the level of immunization inreased (Table 3). Figure 3A shows the number of organisms in the nasal avities and lungs of mie immunized with 5 x 109 ells and subsequently hallenged with 4 x 106 ells (a. 25 LD50) together with data for unimmunized mie. The number of organisms in the nasal avity was 106 to 107 ells in unimmunized mie, whereas the number found in the lungs reahed 109. The multipliation of organisms in the nasal avities and lungs of immunized mie was suppressed. The number of lung organisms was 103 or less on day 20 posthallenge and none were deteted on day 150. In the nasal avity, by ontrast, 104 to 10' ells were deteted even on days 90 to 150 posthallenge. When mie were hallenged with a sublethal dose of 4.8 x 102 ells 14 days after immunization with 5 x 109 ells, the number of organisms in the nasal avity rapidly inreased until day 5, reahing 05, and thereafter gradually dereased; however, organisms were still deteted even 150 days after hallenge (not shown). Fig. 3B shows hanges in the number of organisms in the nasal avities of mie immu- U a - 0' S. 4) V J INFECT. IMMUN. 2 O Days after hallenge FIG. 2. Viable ounts of B. bronhiseptia in brains of unimmunized ontrol mie and mie immunized by the i.p. route with four to five doses of 5 x 109 ells at 1-week intervals. Mie were hallenged i.. 14 days after last immunization with 1.4 x 106 ells of B. bronhiseptia L3. Symbols: 0, unimmunized ontrol mie; 0, mie immunized with five doses; A, mie immunized with four doses.

4 VOL. 41, 1983 EXPERIMENTAL INFECTION WITH B. BRONCHISEPTICA 601 TABLE 2. Survival rate, body and spleen weights, and the ounts of viable B. bronhiseptia in brain, spleen, and heart blood after i.. hallenge in mie with various levels of immunization Immunization Challenge. No. of viable organisms dmmuniatione Challenge Survived/ Day of death Body Spleen per organ in. 10' (viable ells)' ~~~Brain Spleen Heart blood (xdo6ells) (viableells)0 total (day killed) wt (g)' wt (mg) (x 1, x 104 7/10 (20) ND ND ND 200 or x 104 4/ x 106 ND ND x 104 0/ x 107 ND ND a Mie were hallenged by the i.. route with B. bronhiseptia L3 b 14 days after immunization. At 14 days after hallenge. Average of five mie (all the surviving mie in the ase of less than five mie per group). d ND, Not deteted in ml of suspension (<100 ells per organ). nized with three to five doses of 5 x 109 ells and hallenged with 102 ells. In the unimmunized mie, the number of organisms in the nasal avity had reahed 105 by day 3 and was dereasing from day 10 posthallenge. Agglutinin titers at the time of hallenge were highly elevated, being 1:2,560, 1:10,240, and 1:10,240 to 1:20,480 after three, four, and five immunizations, respetively. The multipliation of organisms in the nasal avity was inversely related to the frequeny of immunization. None was deteted in the nasal avities of about half of the mie whih had reeived four to five immunizations, and the average number of organisms was about 103, i.e., 1/100 to 1/1,000 that deteted in the unimmunized mie. However, omplete disappearane of the organisms ould not be ahieved even on days 40 to 60 posthallenge. DISCUSSION Little basi work has been onduted in small experimental animals on the relationship between the prodution of antibodies after immunization with killed B. bronhiseptia vaine and protetion against a live hallenge with B. bronhiseptia. In this study in mie, loser observations and more detailed evaluations have been made of these aspets. Our results show that possible auses of death after i.p. hallenge inlude not only a primary septiemia, already reported in guinea pigs (6), but also an intoxiation due to dermoneroti toxin. The latter is a protein toxin harateristi of the genus Bordetella (4, 9, 16), and the remarkable spleni atrophy it auses was onstantly observed in the present study. Smith et al. (17) showed that mie injeted with killed B. bronhiseptia ells developed levels of agglutinin whih depended upon the vaine strain, immunizing dose, and the antigen used for the agglutination test. In the present study, the elevation of serum agglutinin titers was shown to depend on the frequeny of immunization. When immunized mie were hallenged by the i.p. route with a lethal dose, the speed and rate of learane of the organisms from the spleen and liver in turn depended on the agglutinin titer. Consequently, it an be assumed that in this situation the serum agglutinins were responsible for the protetion. Tanaka TABLE 3. Survival rate, body and spleen weight, and the ounts of viable B. bronhiseptia in nasal avity, lungs, spleen, and heart blood after i.n. hallenge in mie with various levels of immunization Immunization Challenge No. of viable organisms per organ in dmlaose dosllenge Survived/ t0~b Day of death Body Spleen (X106 ells) (viable ells) (akild ~4 avity Lug penblood dos6els (viabe ls)a totab (day killed) wt (g) wt (mg) Nasial dugt Sle bheardt 1, x /10 (20) x 105 NDe (3) ND ND 19 x 102(2) x /10 (20) x 105 ND (1) ND ND 48 x 102(4) x 107 2/10 (20) x 105 ND (1) ND ND 152 x 102(1) x 107 0/ x x x 1io 10' x 106 1/5 (20) x x 102(1) ND ND x 105 5/5 (20) x 105 ND (1) ND ND 124 x 102(4) a Mie were hallenged by the i.n. route with B. bronhiseptia L3 14 days after immunization. b At 14 days after hallenge. Average of five mie (all the surviving mie in the ase of less than five mie per d group). Numbers in parentheses indiate the number of mie. ND, Not deteted in ml of suspension (<100 ells per organ).

5 602 SEKIYA, KAWAHIRA, AND NAKASE INFECT. IMMUN. A 673.-I *a 6 z >. o 4i > to IA A _9 I -0- t *-, 0 > 0 4 o oi 0 4i 0 4i Q -4 4) m -4 (.).0 do.4-4 m > O fo 0-4 S. 0 -i 4) CL l # w f* h * * * #- 4,- 3, Days after hallenge I I S if I WF 0 Days after hallenge FIG. 3. (A) Viable ounts of B. bronhiseptia in nasal avities and lungs of unimmunized ontrol mie and mie immunized by the i.p. route with 5 x 109 ells. Mie were hallenged by the i.n. route 14 days after immunization with 4 x 106 ells of B. bronhiseptia H-16. lungs of unimmunized ontrol mie; 0---0, nasal avities of unimmunized ontrol mie; O-O, lungs of immunized mie; 0---0, nasal avities of immunized mie. (B) Viable ounts of B. bronhiseptia in the nasal avities of unimmunized ontrol mie and mie immunized by the i.p. route with various vaine doses, eah of 5 x 109 ells, at 1-week intervals. Mie were hallenged by the i.n. route 14 days after the last immunization with 102 ells of B. bronhiseptia L3. Symbols: 0, unimmunized ontrol mie; l, immunized with three doses; A, immunized with four doses; 0, immunized with, five doses (19) showed in mie vainated with B. bronhiseptia vaine and subsequently hallenged sublethally with B. bronhiseptia by the i.p. route that the organisms were eliminated within 24 h, suggesting that there was no proliferation. The proliferation of organisms in the ontrol mie was not more than approximately 10 times, and thereafter the number of organisms gradually dereased. It an be assumed, therefore, that phagoytosis by i.p. leukoytes was an effetive means of preventing proliferation in these unvainated mie. Despite the fat that B. bronhiseptia vaine proteted mie against i.p. infetion, the spleen weight was markedly redued even after the omplete elimination of the organisms from the liver and spleen. It seems likely that the toxin ats upon the spleen at an early stage after hallenge (16), before suffiient antitoxin has been produed, and that reovery of normal spleen weight is a slower proess than the omplete elimination of viable organisms from the spleen. The spleens of immunized and unimmunized mie killed by an i.. infetion were atrophied. This was probably due to the diffusion of dermoneroti toxin from the brain to the periphery rather than to a spread of the organisms beause they were not deteted in the spleen and heart blood, although a transient septiemia annot be ruled out. No organisms were deteted in the brain and no spleni atrophy ourred in the surviving immunized mie, showing that the spleni atrophy with i.. infetion of B. bronhiseptia requires a proliferation of the bateria for the systemi effets of the assoiated dermoneroti toxin to be seen. It is likely that the level of immunization (agglutinin titer) against B. bronhiseptia has a bearing on the proliferation of the organisms in the brain, on the inidene of death by enephalitis, and on the pathologial hanges suh as spleni atrophy after i.. hallenge.

6 VOL. 41, 1983 The i.n. route of infetion yielded some interesting results. A small number (a. 100) of B. bronhiseptia organisms was suffiient to effetively olonize the nasal avity, and one established they were diffiult to eradiate. Immunization with killed B. bronhiseptia vaine appreiably suppressed the proliferation of the bateria in the nasal avity and lungs, preventing pneumonia (6) and spleni atrophy. However, despite high agglutinin titers and the omplete elimination of the organisms from the lungs, organisms were still present in the nasal avity even 150 days after the infetion. Similar findings were obtained in mie by Ogata et al. (unpublished data) and in guinea pigs by Nakagawa et al. (10, 11). By ontrast, Ganaway et al. (3) reported that guinea pigs immunized with an adjuvanted killed B. bronhiseptia vaine ould eliminate the organisms from the nasal avity if the hallenge was by natural exposure in the olony. Ganaway et al. obtained their results by ulturing the external nares and nasopharynx and by swabbing the exposed trahea, whereas in our experiments, we quantitated B. bronhiseptia organisms in the nasal avity, throughout the ourse of infetion, by disseting out the whole nasal avity and emulsifying and ulturing it. Thus, it is not lear whether these disrepanies are due to differenes in animal speies, detetion methods, or the vaines used, or also to differenes in the hallenge or immunization methods. However, in mie it seems probable that serum antibodies against surfae antigens are not suffiient to bring about the omplete elimination of infeting organisms from the nasal avity. Our experiments indiate that B. bronhiseptia shows onsiderable variation in its ability to survive, depending on the route of infetion. It is possible that this data may be of more general appliation in analyzing the relationship between host and parasite and the mehanism of pathogenesis and of immunologial defense in baterial infetion, espeially for organisms inhabiting the respiratory trat. These results may be partiularly pertinent to Bordetella pertussis, whih has antigenially similar hemagglutinin (8), dermoneroti toxin, and K-agglutinogen in ommon with B. bronhiseptia (2, 5, 9, 16). Aordingly, we are hoping to larify the mehanisms whih enable the omplete learane of organisms from the respiratory trat, espeially from the nasal avity. ACKNOWLEDGMENTS We thank T. Kasuga for his helpful advie and suggestions and Patrik B. Marley and Alan C. Blaskett (Commonwealth Serum Laboratories, Parkville, Vitoria 3052, Australia) for their useful advie in preparing this manusript. EXPERIMENTAL INFECTION WITH B. BRONCHISEPTICA 603 LITERATURE CITED 1. Cohen, S. M., and M. W. Wheeler Pertussis vaine prepared with phase I ultures grown in fluid medium. Am. J. Publi Health 36: Eldering, G., C. Hornbek, and J. Baker Serologial study of Bordetella pertussis and related speies. J. Bateriol. 74: Ganaway, J. R., A. M. Allen, and C. W. MPherson Pievention of aute Bordetella bronhiseptia pneumonia in a guinea pig olony. Lab. Anim. Care 15: Goodnow, R. A Biology of Bordetella bronhiseptia. Mirobiol. Rev. 44: Goodnow, R. A., C. D. Lehr, F. J. Shade, and J. L. Wlsearver Mouse poteny assay for Bordetella bronhiseptia baterins. J. Clin. Mirobiol. 6: Horie, K., Y. Fujisaki, J. Irisawa, S. Okazaki, and I. Usuba Some aspets of diseases in small laboratory animals. I. Isolation of Bordetella bronhiseptia from the lungs of guinea pigs. Exp. Anim. 9: Kendrilk, P. L., E. B. Nadolski, G. Eldering, and J. Baker Antigeni relationships of Haemophilus pertussis, Parapertussis baillus, and Bruella bronhiseptia as shown by ross protetion tests in mie. J. Bateriol. 66: Munoz, J. J., H. Arai, and R. L. Cole Mouseproteting and histamine-sensitizing ativities of pertussigen and fimbrial hemagglutinin from Bordetella pertussis. Infet. Immun. 32: Munoz, J. J., and R. K. Bergman Bordetellapertussis. Immunologial and other biologial ativities, p In N. Rose (ed.), Immunologial series, vol. 4. Marel Dekker, In., New York. 10. Nakagawa, M., T. Muto, T. Nakano, H. Yoda, K. Ando, Y. Isobe, and K. Imalzumi Some observations on diagnosis of Bordetella bronhiseptia infetion in guinea pigs. Exp. Anim. 18: Nakagawa, M., T. Muto, H. Yoda, T. Nakano, and K. Imaizumi Experimental Bordetella bronhiseptia infetion in guinea pigs. Jpn. J. Vet. Si. 33: Nakgawa, M., H. Yoda, T. Muto, and K. Inaizumi Prophylaxis of Bordetella bronhiseptia infetion in guinea pig by vaination. Jpn. J. Vet. Si. 36: Nakawe, Y Studies on Hemophillus bronhiseptius. III. Differenes of biologial properties between phase I and phase III of H. bronhiseptius. Kitasato Arh. Exp. Med. 30: Nakase, Y., M. Kimura, and K. Shimoda Effiay of an inativating Bordetella bronhiseptia vaine for atrophi rhinitis, p. 8. Proeedings of the International Pig Veterinary Soiety 1976 Congress, Ames, Iowa. 15. Powell, H. M., C. G. Culbertson, and P. W. Ensminger Charoal agar ulture medium for preparing Haemophilus pertussis vaine. Publi Health Rep. 66: Seklya, K., J. J. Munoz, and Y. Nakase Effet of dermoneroti toxin of Bordetella pertussis on the spleen of CFW and C57BL/lOSN mie. Mirobiol. Immunol. 26: Smith, I. M., A. J. Baskervllle, E. Brothwell, and J. Olphant Immunogeniity of killed Bordetella bronhiseptia vaines in the mouse. Res. Vet. Si. 32: Strada, F., and R. Inuina Ueber eine neue Form von infektioeser Lungenkrankheit der Meershweinhen. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 28: Tanaka, J Experimental Baillus bronhiseptius infetion in mie and the effet of immunization on the protetion of mie infeted with Baillus bronhiseptius. I. Saikingaku-zasshi. 519: (In Japanese.) 20. Tanaka, J Experimental Baillus bronhiseptius infetion in mie and effet of immunization on the protetion of mie infeted with Baillus bronhiseptius. II. Intravenous infetion. Saikingaku-zasshi. 560: (In Japanese.)