Production of leukocidin by clinical isolates of Pseudomonas aeruginosa and antileukocidin antibody from sera of patients with diffuse panbronchiolitis

0095-1137/84/110855-05$02.00/0

Copyright © 1984, American Society for Microbiology

Production of Leukocidin by Clinical Isolates of Pseudomonas

aeruginosa

and

Antileukocidin

Antibody from Sera of Patients with

Diffuse

Panbronchiolitis

J. YUZURU HOMMA,l* MOTOHIRO

MATSUURA,'

MITSURU YAMAMOTO,2 YOSHIYUKIKUBOTA,2TOSHIYA HIRAYAMA,3AND IWAO KATO4

The KitasatoInstitute,' Research andDevelopment Center of Hygienic Science, Kitasato University,2 andDepartment of BacterialInfection, Institute of Medical Science, University of Tokyo,3 Tokyo 108, and SecondDepartment of

Microbiology, School of Medicine, Chiba University, Chiba280,4Japan Received 30May 1984/Accepted8August 1984

The ratio ofleukocidin-producing strains toclinical isolates ofPseudomonas aeruginosa was investigated togetherwith the productionof protease, elastase, andexotoxin A. We also examined whether these strains contain thecommon antigen which resides in the cell wall. By usingtheagar geldiffusion testwith specific antisera, we found that 87 of 90 (96.7%) ofclinical isolates produced leukocidin. Protease, elastase, and exotoxin Awerealsoproducedathighpercentages.Thecommonantigenwasfoundtoexistinallstrains.Next, toestimate antileukocidin antibody in the seraofpatients, weusedan enzyme-linkedimmunosorbent assay

with horseradish peroxidase-protein A. The sera of 39 patients with diffuse panbronchiolitis (DPB) were investigatedfor antileukocidinantibody.Themeanantileukocidin titer in theseraof17 DPBpatientswhowere notinfected withP.aeruginosaand 5 DPBpatientswhoweretransiently infected with the bacteriawasabout thesame asthemeanantileukocidin titer intheseraof 11healthy controls, whereasthemeanantileukocidin titer intheseraof 17 DPBpatientswhowerepersistentlycolonizedwassignificantly higherthanthat inhealthy

controls. These results indicate that leukocidin wasproducedatthe localsite of infection in DPBpatients. There are many reports on the roles of exotoxin A and

exoenzymesof Pseudomonasaeruginosainthe

pathogenici-ty of P. aeruginosa infections (5, 15). Leukocidin was characterized by Scharmann (18-21) and Lutz (9) as a cytotoxic protein distinct from other P. aeruginosa toxins.

However, only 4 of110 strains tested produced detectable amounts of the protein. None of the reports on the role of leukocidin in the pathogenicity ofpseudomonal infections except Scharmann's have suggested that the neutropenia

found inpseudomonal sepsis iscausedby the action of this

toxin.

In this paper, we investigated (i) the ratio of

leukocidin-producing strains to clinical isolates of P. aeruginosa by using an agar gel diffusion test with rabbit antileukocidin serumand(ii)antileukocidin antibodyin theseraofpatients with diffuse panbronchiolitis (DPB) (see reference 4) by enzyme-linked immunosorbent assay (ELISA), compared with that in normal human sera.

MATERIALS AND METHODS

Strains. Leukocidin-producingstrain 158wasprovided by

W. Scharmann, University ofGiessen, Giessen, West Ger-many. A total of 69 serotyping strains ofP. aeruginosa, including 22 strains from B. Lanyi, National Institute of

Public Health, Budapest, Hungary; 7 strains from M. W.

Fisher, Parke, Davis &Co., Detroit, Mich.; 11strains from

I. Habs, Hygiene-Institut der Universitat Heidelberg,

Hei-delberg, West Germany; 14 strains from E. Verder and J.

Evans,National Institute ofAllergyandInfectiousDiseases, Bethesda, Md.;and 15 strainsfromJ.Y.H.,andatotal of 21 clinical isolates of P. aeruginosa donated by S. Tanimoto,

Toranomon Hospital, Tokyo, Japan, and K. Inatomi,

Jun-tendo University, Tokyo, Japanwere tested for leukocidin production. Atotal of 30P. aeruginosastrains isolated from

*Corresponding author.

riverwaterand donated byH.Kodama, Toyama Institute of Health, Toyama, Japan, were also tested for leukocidin production.

Antigens and antisera. Crystalline leukocidin (3), highly purified exotoxin A(23)and aproteinaceous common anti-gen(designated asOEP) (24)wereused. Crystalline alkaline protease and elastase were obtained from Nagase Ltd., Osaka, Japan. The two enzymes were prepared by the

methods of Morihara (10)and Moriharaetal. (11).

Antileukocidin sera were obtained from three rabbits immunized with a mixture of leukocidin and Freund com-plete adjuvant. Each rabbitwassubcutaneously immunized three times at intervals of 4 weeks. A total of 110 ,ug of

leukocidin solutions (three solutions containing 10, 50, and

50 ,ug of leukocidin mixed with equal volumes of Freund

complete adjuvant) was administered. At 2 weeks afterthe lastimmunization, the rabbitswere sacrificed, and theirsera were separated. Antileukocidin titers of theserawerefound to bepositiveata10,000-fold serumdilution byanELISA. Antiserum neutralized the cytotoxic activity of leukocidin (I. Kato, personal communication).

Antiprotease serum, antielastase serum, antiexotoxin se-rum, and anti-OEP serum were obtained by methods de-scribedpreviously (6, 22, 25). Allthe antiserawerefoundto bepositiveatserumdilutionsof5,120to10,240byapassive

hemagglutination test with sheep erythrocytes coated with protease,elastase, exotoxin, orOEP.

Sera of DPB patients were kindly provided by S. Tani-moto,ToranomonHospital, and K. Inatomi, Juntendo Uni-versity, and preserved at -70°C.

Agar cultures for detectingleukocidin production. Tryptic

soy broth (10 ml; Difco Laboratories, Detroit, Mich.) con-taining1%(vol/vol) glycerol, 5% (vol/vol) sodium glutamate,

and 1.5% (wt/vol) agarose L was poured into a petri dish (diameter, 90 mm).

ELISA.Polystyrene microtiter plates (Flow Laboratories, 855

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Inc., McLean, Va.) were used. Wells of microtiter plates

were filled with 0.2-ml volumes of antigen (10 ,ug/ml) pre-pared in 0.05 M carbonate buffer (pH 9.8). The plates were incubatedovernight in a humiditychamber at 4°C and then washed three times with 0.1 MTris-hydrochloridebuffer (pH 7.6). Tominimizenonspecificadsorption, we filled each well with 0.1 M Tris-hydrochloride buffer (pH 7.6) containing 0.85% NaCl, 1% bovine serum albumin (Sigma Chemical

Co., St. Louis, Mo.), and5%fetal calf serum and incubated the plates at 37°C for 1 h. After the masking solution was

discarded, dilutedsera were added in 0.2-ml volumes to the wells. After beingincubated at 37°C for 1 h, the plates were washed three times with 0.1 MTris-hydrochloridebuffer (pH 7.6).

Apositivecontrol serum was obtained from DPB patients with a prolonged history of colonization (more than 6

months) ofsputum by P. aeruginosa. A negative control

serumwasobtainedfrom healthy humans with nohistory of DPB. These sera were diluted with 0.1 MTris-hydrochloride buffer(pH7.6)containing 1%bovine serum albumin and 1% Tween 20.

Horseradishperoxidase (HRPO)-proteinA conjugate was prepared by the method of Nakane and Kawaoi (12) with HRPO (GIBCO Laboratories, Grand Island, N.Y.) and

protein A (Sigma Chemical Co.). Theconjugate solution (1

jiglml)

(pH 7.6) containing 0.85% NaCl and 1% bovine serum albumin and was added to all wells in 0.2-ml volumes. The plates wereincubated at roomtemperaturefor 1 h in thedark andthen washed three times with 0.1 MTris-hydrochloride

buffer (pH 7.6). The substrate, 0.012%H202, and 0.16%

0-phenylendiamine hydrochloride prepared in 0.1 M

phos-phate buffer (pH 6.0) were added to each well in 0.2-ml volumes. After 10 min of incubation at roomtemperature in the dark, the reaction wasstopped by the addition of 0.05 ml

of4 N H2SO4. Absorbance values for the content ofeach well weremeasured at 492 nm with aspectrophotometerfor

microplates(MTP-2; Corona Electric Inc., Ibaragi, Japan). Asthe absorbancevaluesofseradilutedto 1:10 inrelation topositive and negative controlswere0.40 and 0.13,

respec-tively, and as thedeviationofeachsamplewasfound tobe

small when a 1:10 serum dilution was applied, the serum

dilutionused in theroutinetest was 1:10.

RESULTS

Agar gel diffusion test with leukocidin and antileukocidin

sera.TheOuchterlonymethod(14) was used. Serial

concen-trations of leukocidin solubilized in 0.05 M phosphate-buffered saline wereexaminedtofindtheproper

concentra-tion ofthe antigen forthe agargel diffusiontest (Fig. 1). It was found that antileukocidin serum reacted with all the

leukocidinsolutions containing 5, 10, and100Fxg of

leukoci-dinper ml. As theprecipitinline of 5 ,ug/mlwasobscure, 10 ,ugof leukocidinper ml was found tobethebest

concentra-tion and was adopted foruse in theprecipitintest.

We next investigated whether the agar gel diffusion test would be affected by protease or elastase produced by P. aeruginosa. One milliliter of 0.05 M phosphate-buffered saline (pH 7.2) containing 20

pg

added to an equal volume (1 ml) of protease or elastase solutioncontaining0.1 MTris buffer (pH 7.4)with 100 mM

sodium acetate, 20 mM calciumchloride, and 0.5 mM zinc

chloride. Theenzymeconcentrations werebroughtto2, 20,

and 200,ug/ml. After beingincubated at37°C for1 hand4°C overnight,themixed antigen solutionsweresubjectedto the agar gel diffusion test with antileukocidin serum. As the

control, 10

pLg

leukocidin

phosphate-buffered saline (pH 7.2) was used. No difference was ob-served between the precipitin patterns of the antigen mix-tures and those of the control solutions. It was concluded that theagar gel diffusion test is not affected by protease or elastase produced by P. aeruginosa.

Serological specificity of leukocidin. The serological speci-ficity of leukocidin was examined by using the agar gel diffusion test.Antileukocidin serum was poured into a center well; into six peripheral wells located at radii of 2 mm from the center well and 2 mmfrom each other,different concen-trations (100 to 10 ,ug/ml) of the test antigen were poured. After the plate was incubated at room temperature over-night, the reaction was determined. It was found that antileu-kocidin serum reacted with leuantileu-kocidin but not with the other four antigens (protease, elastase, exotoxin, or OEP). The serological specificity of leukocidin was thus proved. Re-garding the other four antisera, it was also proved that the precipitin test was positive only in homologous antigen-antiserum systems and was always negative in heterologous antigen-antiserum systems. These tests confirmed that all five antisera possessed serological specificity.

Production of leukocidin by P. aeruginosa clinical isolates. To detect theproduction of leukocidin as well as of protease, elastase, and exotoxin A and to detect OEP, we inoculated a test strain on one side of a petri dish with an agar culture. The dish was incubated at 37°C for 2 days and then exposed tochloroform vapor. The purified leukocidin and the antileu-kocidin serum were then placed into two wells of the plate, which was incubated at room temperature overnight. If the strain produced leukocidin, a precipitin line appeared after the dish was incubated at 25°Covernight. Figure 2A showed aprecipitin line against leukocidin from a culture fused into a precipitin line against crystalline leukocidin. In the case of protease, elastase, and exotoxin A, a similar phenomenon wasobserved.

To detect the four kinds of metabolites and OEP at the sametime, we cultivated and killed (withchloroform vapor) atest strain as described above. Five wells were made5mm from thegrowing culture and filled with five kinds of antisera (Fig. 2B). The precipitin reaction was determined after the dish was incubated at 25°C overnight. Of the 21 clinical isolates and 69 serotyping strains, a total of 87 strains were found to produce leukocidin. Protease, elastase, and exotox-in A were also produced at high percentages. OEP was found toexist in all strains. These results are shown in Table 1. P. aeruginosa isolated from environmental (river water)

FIG. 1. Agar gel diffusion test with leukocidin and rabbit antileu-kocidin serum. Serial concentrations of leukocidin solubilized in 0.05 Mphosphate-buffered saline were examined to find the proper concentration of the antigen for the agar gel diffusion test. A clear precipitin line was observed at antigenconcentrations of 10 and 100 ,ug/ml.

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1.0

r-0%~

z

(,, 0.;

0

(A

B

6

FLI. 2.

diffuslon

of

isolate

wasinoculatedononeside ofapetri dish, whichwasthenincubated

at37°C for2daysandexposedtochloroformvapor.Awellwasthen madeatadistance ofca. 5mmfrom thegrowingculture and filled with an antiserum for the agar gel precipitin test. A positive or

negative precipitinreactionwasdetermined after thepetridishwas

incubated at 25°C overnight. (A) Precipitin line against leukocidin fromaculture fusedintoaprecipitinlineagainst crystalline leukoci-din. 1, Antileukocidin serum;2,leukocidin (10 ,ug/ml); 3, growing culture ofanisolate.(B)Positiveprecipitinreactionsagainstall four metabolites and OEP. 1, Antileukocidin serum; 2, antielastase serum; 3, antiprotease serum; 4, antiexotoxin serum; 5, anti-OEP serum;6, growingculture ofan isolate.

sourcesproduced leukocidin aswell asother metabolites at

high percentages and alsowas foundtocontain OEP. Antileukocidin antibody in sera ofDPB patients. To esti-mate antileukocidin antibody in sera of DPB patients, we usedan ELISA with theHRPO-protein A conjugate. Sera of 39 DPB patients were investigated for antileukocidin anti-body. Antibody titers in DPB patients and in healthy con-trols are shown in Fig. 3. Mean antibody titers were com-pared withatwo-tailed Student'sttest.Meanantibody titers in DPB patients not infected with P. aeruginosa (0.12 +

0.08) and in DPB patients transiently infected with the bacteria (0.17 + 0.08) were not significantly different from the mean antibody titer in healthy controls (0.13 + 0.08),

whereasthemeanantibody titer in DPB patients persistently colonized with P. aeruginosa (0.52 + 0.26) wassignificantly

Heal thy control

0

0

Uncolonized Transiently

colonized

0

0-0@

I.

I

0

Persi stently colonized

Diffuse panbronchiolitis

FIG. 3. Antibody againstleukocidin in the seraofDPBpatients and healthy controls. Antileukocidin antibody in thesera ofDPB

patients was estimated by an ELISA with the HRPO-protein A conjugate. The concentration of leukocidin and of the HRPO-protein Aconjugatewas 1 ,ug/ml.Thedilutionof the tested serum was 1:10. Barsindicatemean ± standard deviation.

higher (P < 0.001) than that in healthy controls. These results indicate thatleukocidin is producedatthelocal siteof infection in DPB patients.

DISCUSSION

In a previous report (17a), the percentage of clinical isolatesofP. aeruginosa producing leukocidinwasfoundto

beverylow(3.6%). Thepresentinvestigation proved, byuse

ofan agargel diffusion test, that almost all clinical isolates produce leukocidin. Thisdiscrepancymaybeascribedtothe methods used. In the former, leukocidin production was confirmed by the destruction of leukocytes caused by the culture filtrateof theisolate tested. I.Kato and T. Hirayama

confirmed that the rabbit antileukocidin serum used in our experiments blocked completely the cytotoxic activity of the

TABLE 1. Production of leukocidin, protease, elastase, and exotoxin byP. aeruginosa and presence ofOEP in test strains isolated from clinical and river watersamplesa

No.(%)ofstrainsproducing: Isolates(n)

Leukocidin Protease Elastase Exotoxin OEP

Clinical

Serotyping strains (69) 68 57 55 68 69

(96.7) (82.2) (83.3) (98.9) (100)

Freshly isolated strains (21) 19 17 20 21 21

Riverwater(30) 30 (100) 22(73.3) 29 (96.7) 30 (100) 30 (100)

a Examinedby theagargel diffusiontest(Fig.2).

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purified leukocidin (personal communication). A single pre-cipitin line against leukocidin from a culture fused into a precipitin line against crystalline leukocidin (Fig. 2A). All 117 strains found to produce leukocidin formed a clear, single precipitin line against rabbit antileukocidin serum. These results suggest that there was no cross-reactive pro-tein other than leukocidin in the culture.

As for the antisera used, there was no serological cross-reaction against leukocidin, protease, elastase, exotoxin A, or OEP in the agar gel diffusion test. It has been also reported by Takeshi and Homma (22) that each of the antisera exhibited no serological cross-reaction against pro-tease, elastase,exotoxin A, or OEP in the passive hemagglu-tination test.

It was reported by Pollack et al. (16), Bjorn et al. (1), and Sanaiet al. (17) that most clinical isolates producedexotoxin A at a high percentage, whereas isolates from hospital environments produced exotoxin A at a low percentage(17). Contrary to the above results, isolates from river water sources were found to produce exotoxin A as well as protease and elastase at a high percentage. Okuda (13) also reported that isolates from river water sources produced

elastase at a high percentage (97.6%). Okuda (13) and Kodama and Kubo (8) foundthat serogroupsof isolates from the lower part of the river (downstream) were similar to

those of clinical isolates in the area. On the basis of these results, they suggested thattheisolates from riverwatermay havesome relationship tohumanP.aeruginosaorigins.This

is a problemwhich needs further investigation.

The present investigation proved that almost all clinical

isolatesproduceleukocidin as well asexotoxin A, protease,

andelastase andthat the meanantileukocidin titer inthesera

ofpersistently colonized DPB patientsissignificantly higher

than that in the sera of noncolonized and transiently

colo-nized DPB patients and healthy humans. Cho et al. (2) investigated antibody titers against protease, elastase, and

OEPbyapassivehemagglutinationtest andimmunoglobulin

E levels in the sera of 29 cystic fibrosis (CF) patients and

found highpassive hemagglutination titers against protease, elastase, and OEP and high immunoglobulin E levels inthe

seraof the CF patients. Jagger et al. (7) notedmoreprecisely byusinganELISA that levelsof antibodiesagainstexotoxin

A, protease, and elastase in colonized and infected CF patientswerehigher than levelsinnoncolonized CFpatients and control subjects without CF. These results suggestthat

persistently colonized patients may experience more

pro-longed exposure to leukocidin as well as to exotoxin A,

protease, elastase, and OEP produced byP. aeruginosa in

sputumand thattheseextracellularmetabolitesareabsorbed

through the mucous membrane of the trachea to elicit antibody responses.

As the cytotoxicity of leukocidin has been found to be verystrong, theroleofleukocidinin the

pathogenicity

aeruginosa infections should be clarified.

ACKNOWLEDGMENTS

We thank W. Scharmann (University ofGiessen) forthe gift of strain 158, S. Tanimoto (Toranomon Hospital) and K. Inatomi (JuntendoUniversity) forclinicalisolates, andH.Kodama(Toyama Institute of Health) for environmental isolates. Serotype strains

werekindly given to J.Y.H. between 1958 and 1963 andpreservedin hislaboratory.

LITERATURE CITED

1. Bjorn, M. J., M. L. Vasil, J. C. Sadoff, and B. H.Iglewski.1977. Incidence of exotoxin production by Pseudomonas species. Infect. Immun. 16:362-366.

2. Cho,J. Y., Y. H. Oh,C.Abe, J. Y.Homma, M. Usui, and T. Matsuhashi. 1978.IgE antibody productiontoexoenzymesand common antigen (OEP) of Pseudomonas aeruginosa. Jpn. J. Exp.Med. 48:491-496.

3. Hirayama, T.,I. Kato,F.Matsuda, and M. Noda.1983. Crystal-lization and someproperties of leukocidin from Pseudomonas aeruginosa. Microbiol. Immunol. 27:575-588.

4. Homma, H., A. Yamanaka, S.Tanimoto,M.Tamura, Y. Chiji-matsu,S. Kira, and T. Izumi. 1983.Diffusepanbronchiolitis.A disease ofthe transitionalzoneof thelung. Chest. 83:63-69. 5. Homma, J. Y. 1982.Perspectivesonthedevelopment ofa new

vaccine against Pseudomonas aeruginosainfection,p.389-431. InC. S. F. EasmonandJ.Jeljaszewicz (ed.), Medical Microbi-ology, vol. 1.Academic Press, Inc., London.

6. Homma, J. Y., T. Tomiyama, H.Sano,Y.Hirao, and K. Saku. 1975.Passivehemagglutination reactiontestusing formalinized sheep erythrocytes treated with tannic acid and coated with protease orelastase ofP.aeruginosa.Jpn. J.Exp. Med. 45:361-365.

7. Jagger, K. S., D. L. Robinson, M. N. Franz, andR.L. Warren. 1982. Detection by enzyme-linked immunosorbent assays of antibody specific for Pseudomonasproteasesand exotoxinAin serafrom cystic fibrosis patients. J. Clin. Microbiol. 15:1054-1058.

8. Kodama, H., and Y. Kubo. 1981. Serovar distributions of

Pseudomonasaeruginosafromtheenvironmentand theclinical specimen, p.22-24. In J. Y. Homma (ed.),Proceedings ofthe 15th annual meeting of the Japan Pseudomonas aeruginosa Society. TokyoUniversity Press, Tokyo.

9. Lutz, F. 1979. Purification ofacytotoxic protein from Pseudo-monasaeruginosa.Toxicon 17:467-475.

10. Morihara, K. 1963. Pseudomonas aeruginosa proteinase. I. Purification and general properties. Biochim. Biophys. Acta. 73:113-124.

11. Morihara, K., H. Tsuzuki, T. Oka, H. Inoue, and M. Ebata. 1965. Pseudomonas aeruginosaelastase. Isolation,

crystalliza-tion, andpreliminary characterization.J.Biol. Chem. 240:3295-3304.

12. Nakane, P. K., and A. Kawaoi. 1974. Peroxidase-labeled anti-body.A newmethodofconjugation.J. Histochem.Cytochem. 22:1084-1091.

13. Okuda,K.1981.Serologicalgroupingand elastaseproductionof 543 Pseudomonasaeruginosa strains fromriverwater, p. 6.In J. Y. Homma (ed.),Proceedings ofthe 15th annualmeetingof the Japan Pseudomonas aeruginosa Society.TokyoUniversity

Press,Tokyo.

14. Ouchterlony, 0.1962.Diffusion-in-gelmethodsfor

immunologi-calanalysis. Prog. Allergy. 6:30-54.

15. Pavlovskis, 0. R., and B.Wertlind. 1982. Pseudomonas aeru-ginosatoxins,p.97-128.In C. S.F. Easmonand J.

Jeljaszewicz

(ed.), Medical microbiology, vol. 1. Academic Press,

Inc.,

London.

16. Pollack, M., N. S. Taylor, and L. T. Callahan, III. 1977. Exotoxinproduction by clinical isolates of Pseudomonas aeru-ginosa. Infect. Immun. 15:776-780.

17. Sanai, Y., K. Takeshi, J. Y. Homma, and H. Kamata. 1978. Production ofexotoxin, proteaseand elastase of Pseudomonas aeruginosa strains isolated from patients and environmental specimens.Jpn. J. Exp. Med. 48:553-566.

17a.Scharmann, W. 1976. Formation and isolation of leukocidin from Pseudomonasaeruginosa.J.Gen.Microbiol. 93:283-291. 18. Scharmann,W.1976. Purification andcharacterization of leuko-cidin from Pseudomonas aeruginosa. J. Gen. Microbiol. 93:292-302.

19. Scharmann, W. 1976. Cytotoxic effects of leukocidin from Pseudomonas aeruginosa on polymorphonuclear leukocytes

from cattle. Infect. Immun. 13:836-843.

20. Scharmann, W. 1976. Interaction of purified leukocidin from Pseudomonas aeruginosa with bovinepolymorphonuclear leu-kocytes. Infect. Immun. 13:1046-1053.

21. Scharmann, W., F. Jacob, and J. Prostendorfer. 1976. The cytotoxic action ofleukocidin from Pseudomonas aeruginosa

on human polymorphonuclear leukocytes. J. Gen. Microbiol. 93:303-308.

on February 8, 2020 by guest

http://jcm.asm.org/

22. Takeshi, K., and J. Y. Homma.1978. Passive hemagglutination reaction using formalinized sheep erythrocytes treated with tannic acid and coated with exotoxin of Pseudomonas

aerugin-osa.Jpn. J. Exp. Med. 48:497-501.

23. Takeshi, K., J. Y. Homma, I. Kato, and H. Saito. 1977. Skin necrotizing property of Pseudomonas aeruginosa exotoxin. Jpn. J. Exp.Med. 47:323-325.

24. Tanamoto, F., C.Abe, J. Y. Homma, K.Kuretani, and A. Hoshi.

1978. Compound possessing antitumor andinterferon-inducing activities derived from the commonantigen (OEP) of Pseudo-monasaeruginosa. J. Biochem.83:711-718.

25. Tomiyama, T., J. Y. Homma, C. Abe, and M. Yoichi. 1973. Passive hemagglutination reaction using sheep erythrocytes treated with tannic and coated with protein moiety of the endotoxin of Pseudomonas aeruginosa. Jpn. J. Exp. Med. 43:185-189.

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