Identification and Characterization of the Pasteurella

Transcription

1 INFECTION AND IMMUNITY, OCt. 1987, p /87/ $02.00/0 Copyright 1987, Americn Society for Microbiology Vol. 55, No. 10 Identifiction nd Chrcteriztion of the Psteurell hemolytic Leukotoxin YUNG-FU CHANG,' RY YOUNG,' DAVID POST,2 AND DOUGLAS K. STRUCK2* Deprtment of Biochemistry nd Biophysics, College of Agriculture,' nd Deprtment of Medicl Biochemistry nd Genetics, College of Medicine,2 Texs A&M University, College Sttion, Texs Received 6 April 1987/Accepted 26 June 1987 The identifiction nd chromtogrphic chrcteriztion of the leukotoxin of Psteurell hemolytic is described. The toxin, which hs n pprent ntive moleculr weight of greter thn 400,000 s judged by gel exclusion chromtogrphy, hs 105-kilodlton (105K) polypeptide s its mjor protein component. The proteolytic degrdtion of the 105K polypeptide could be correlted with the loss of toxin ctivity in ging cultures of P. hemolytic. Antiser rised ginst purified 105K polypeptide neutrlized toxin ctivity. A 3.9-kilobse-pir frgment of the P. hemolytic genome cloned into plsmid vector resulted in the production of intrcellulr toxin in Escherichi coli host cells. The restriction mp of this clone shows significnt overlp with the mp of previously reported leukotoxin clone (R. Y. C. Lo, P. E. Shewen, C. A. Strthdee, nd C. N. Greer, Infect. Immun. 50: , 1985). Finlly, ntiser rised ginst the 105K species lbeled the P. hemolytic cell surfce in nonuniform, punctte mnner. Psteurell hemolytic is the pthogenic gent in pneumonic psteurellosis, which is principl fctor in the morbidity nd mortlity ssocited with the complex syndrome clled shipping fever. This orgnism secretes toxin which kills the leukocytes of cttle, gots, nd sheep but not those of nonruminnts (5, 12). Since phgocytic cells re considered to be mjor mechnism of host defense ginst respirtory infections, the P. hemolytic leukotoxin is likely to be mjor virulence fctor. There hve been severl reports of the identifiction of the P. hemolytic toxin from culture superntnts. Himmel et l. (10) purified protein with ntive moleculr weight of 150,000 s judged by gel filtrtion nd described its dissocition into 20,000- nd 50,000-moleculr-weight components in the presence of sodium dodecyl sulfte (SDS). However, their most purified preprtions killed less thn 1% of bovine lveolr mcrophge cells fter 6-h exposure by using trypn blue exclusion. Bluyut et l. (2) climed, on the bsis of ultrfiltrtion, tht the leukotoxin hs ntive moleculr weight greter thn 300,000 but did not chrcterize the mteril t the moleculr level. Lo et l. (13) reported the cloning of P. hemolytic DNA frgment, the expression of which yielded leukotoxin ctivity in Escherichi coli cell lystes. Mosier et l. (17) reported tht the ntive cytotoxin hs moleculr weight of 160,000 s judged by gel filtrtion chromtogrphy. This mteril yielded number of distinct polypeptide species when subjected to SDS-polycrylmide gel electrophoresis (SDS-PAGE). In this report, we demonstrte tht the toxin hs single polypeptide component, with n pprent moleculr weight of 105,000, s judged by SDS-PAGE, which hs primry proteolytic brekdown product with moleculr weight of 95,000 both in P. hemolytic culture superntnts nd within E. coli cells crrying the cloned toxin gene. MATERIALS AND METHODS Bcteril strins, medi, nd culture conditions. Representtive P. hemolytic strins from ech of the 15 * Corresponding uthor. serotypes (7) were obtined courtesy of E. L. Biberstein, University of Cliforni, Dvis. Biotype A, serotype 1 strins were obtined from the Texs Veterinry Dignostic nd Lbortory System in College Sttion nd Amrillo, Tex. P. hemolytic cultures were grown in brin hert infusion (BHI; Difco Lbortories), Dulbecco miniml essentil medium (DMEM) supplemented with 10% fetl clf serum (FCS), or dilyste (DBHI) obtined by dilyzing 10-fold-concentrted BHI ginst 9 volumes of distilled wter. LB-Amp ws Luri broth (16) supplemented with mpicillin (50,ug/ml). Miniml glycerol medium ws M9 miniml medium (16) with 0.2% glycerol s crbon source. SDS-PAGE nd Western blotting. SDS-PAGE ws performed s previously described (1). Western blotting (immunoblotting) ws performed exctly s described by Towbin et l. (22). Immunorective bnds were visulized with second ntibody conjugted to horserdish peroxidse (9). Leukotoxin ssy. The continuous bovine lymphom cell line BL-3 obtined from G. Theilen, University of Cliforni, Dvis, ws used to estimte leukotoxin ctivity. BL-3 cells undergo chrcteristic morphologicl chnges upon exposure to ctive leukotoxin (Fig. 1). In stndrd ssy, 106 cells were incubted with toxin smples (less thn 50,ul) for 1 hour t 37 C in 1 ml of growth medium, which ws Leibovitz L-15 Medium with L-glutmine (Gibco Lbortories) supplemented with 10% FCS. It ws observed tht certin lots of FCS were inhibitory to toxin ctivity. Some ssys were performed in phosphte-buffered sline (8 g of NCl, 0.2 g of KCI, 1.15 g of N2HPO4, 0.2 g of KH2PO4, nd 1 g of glucose per liter, supplemented with 0.5% bovine serum lbumin nd 1 mm CC12). The clcium ws found to be required for leukotoxin ctivity t low concentrtions of toxin nd could not be replced by mgnesium, mngnese, or zinc. One unit of toxin ctivity is defined s the miniml mount of toxin, s determined by seril dilution, required to produce the morphologicl chnge in >95% of the input BL-3 cells. This simple visul ssy correltes well with more tedious ssys bsed on the killing of freshly isolted bovine mcrophges, leukocytes, nd neutrophils (2, 4, 18), s well s with similr ssy bsed on neutrl red uptke of BL-3 cells described by Greer nd Shewen (8). 2348

2 VOL. 55, 1987 FIG. 1. Visul ssy of leukotoxin ctivity by using bovine lymphom (BL-3) cells. Cells were incubted in the bsence (A) or presence (B) of leukotoxin s described in Mterils nd Methods. Phse-contrst microgrph mgnifiction, x250. Source of neutrlizing ntibodies. Antibodies from cttle experimentlly infected with live P. hemolytic were obtined from R. Lon, Deprtment of Veterinry Microbiology nd Prsitology, College of Veterinry Medicine, Texs A&M University. Antibodies were lso rised in New Zelnd White mle rbbits immunized with Formlin-killed P. hemolytic. In ddition, ntibodies specific for the 105-kilodlton (105K) polypeptide species secreted by P. hemolytic were rised in New Zelnd White mle rbbits by immuniztion with mteril purified by preprtive SDS- PAGE. Gel slices contining the 105-kilodlton bnd were directly emulsified with Freund djuvnt nd injected subcutneously into rbbits. Immunologicl techniques. Immunoprecipittion of culture superntnts nd French presstes nd nlysis of SDSpolycrylmide gel were done s previously described (15). Immunoprecipittion of whole-cell extrcts were done essentilly s described by Stder et l. (20). Briefly, 1 ml of sturted culture of cells ws centrifuged for 2 min in n Eppendorf Microfuge (Beckmn Instruments, Inc.), nd the pellet ws suspended in 50,u1 of 1% SDS-1 mm EDTA (ethylenediminetetrcetic cid), ph 7.8. This suspension PASTEURELLA HAEMOLYTICA LEUKOTOXIN 2349 ws boiled for 1 min, supplemented with 0.65 ml of TSET buffer (50 mm Tris, ph 8.0, 150 mm NCl, 1 mm EDTA, nd 2% Triton X-100), nd centrifuged gin for 10 min t room temperture in the microfuge. The insoluble pellet ws removed with toothpick, nd the superntnt ws immunoprecipitted s described bove. Indirect immunofluorescent stining of P. hemolytic ws performed by using fresh, logrithmic-phse cells grown in BHI. The cells from 50,ul of culture were wshed three times in sterile 0.15% NCl. After the lst wsh, the cells were resuspended in 50,ul of undiluted ntiserum nd incubted t 37 C for 30 min. After incubtion, the cells were wshed three times with PBS. For stining, the cells were resuspended in buffer contining fluorescein-conjugted got nti-rbbit immunoglobulin G (hevy nd light chin specific; Cppel Lbortories) nd incubted t 37 C for 0.5 h. After the incubtion, the cells were wshed with phosphte-buffered sline three times nd were immobilized on n gr-coted microscope slide. Microscopy ws performed with Leitz Dilux 20 microscope equipped with phse-contrst optics nd epiillumintion for fluorescence. Preprtion of crude leukotoxin. P. hemolytic biotype A, serotype 1 ws grown in DBHI to n A550 of 4, nd the culture superntnt ws collected by low-speed centrifugtion. The superntnt ws concentrted 10-fold by ultrfiltrtion by using n Amicon DC-2 hollow-fiber pprtus with moleculr weight cutoff of 30,000. Tris bse (20 mm, 2 liters) djusted to ph 8.0 with HCI ws dded, nd the mixture ws concentrted 20-fold by ultrfiltrtion. This step ws necessry to chieve the slt nd ph requirements of DEAE ion-exchnge chromtogrphy, since dilysis t this stge resulted in the loss of toxin ctivity. The concentrted superntnt ws subjected to ultrcentrifugtion t 117,000 x g for 1 h t 4 C to remove insoluble mteril. This preprtion contined typiclly 5,000 U/ml nd ws used s the crude toxin preprtion in ll chromtogrphy experiments. Cloning of toxin gene. P. hemolytic chromosoml DNA ws prepred by the method of Silhvy et l. (19) nd prtilly digested with Su3A. The digest ws frctionted by sedimenttion in neutrl sucrose (14), nd frctions contining 3- to 10-kilobse-pir frgments, s judged by grose gel electrophoresis, were pooled nd concentrted by lcohol precipittion to finl level of 100 p.giml. The omeg-complementing plsmid phg165 ws chosen s the librry vector. Plsmid vector phg165 (10,ug) (21) ws cleved with BmHI nd treted with lkline phosphtse to remove terminl phosphtes. After phenol extrction nd concentrtion by ethnol precipittion, the vector DNA ws mixed with the 3- to 10-kilobse-pir P. hemolytic DNA t TABLE 1. Antiserum source Neutrliztion of leukotoxin ctivity Neutrliztion t ntibody dilution: 1 1:2 1:4 1:8 1:16 1:32 1:64 Cow Rbbit ± + + Rbbit (preimmune) + ND ND ND ND ND ND smple of prtilly purified toxin (100 U) ws mixed with 10-,ul of serilly diluted ntiserum obtined from experimentlly infected cttle or from rbbit immunized with the 105K polypeptide which hd been purified by preprtive SDS-PAGE. After 1-h incubtion t 37 C, 106 BL-3 cells in 1 ml of L-15 growth medium ws dded to ech mixture nd toxin ctivity ws ssessed s described in Mterils nd Methods. +, >95% of the BL-3 cells showed morphologicl chnges induced by leukotoxin; -, complete neutrliztion of leukotoxin; ND, not done. A 1-pIU

3 2350 CHANG ET AL. INFECT. IMMUN * 0 W11* 68-..: v I z9 68~~~~~~~~~~~~~~~~~~~~~~~~~~~!*A _-...4 z FIG. 2. Immunoprecipittion of culture superntnts from different P. hemolytic serotypes. P. hemolytic cultures were grown in BHI medium supplemented with 10% FCS. Lte logrithmic-phse superntnt (300,ul) ws immunoprecipitted with 10,ul of bovine ntiserum nd nlyzed on polycrylmide gel with silver stining s described in Mterils nd Methods. Moleculr size is given in kilodltons. Lnes: 1 nd 25, moleculr weight stndrds; 2, control immunoprecipittion by using 300,ul of growth medium; 3 to 17, P. hemolytic serotypes 1 to 15; 18 to 24, different clinicl field isoltes of P. hemolytic biotype A, serotype 1. The mjor diffuse bnd t pproximtely 50 kilodltons is the immunoglobulin G hevy chin dded in the immunoprecipittion. molr rtio of 1:5 nd treted with T4 DNA ligse for 16 h t 18 C. The ligtion mixture ws trnsformed into lphcomplementing E. coli TB1 hsdr (11), nd trnsformnts were selected on LB-Amp with 100,ug of X-Gl (5-bromo- 4-chloro-3-indolyl-p-D-glctopyrnoside) per ml. Trnsformnts were obtined t pproximtely 1,000 colonies per pg of input DNA, pproximtely 80% of which were colorless nd thus contined insert DNA. Immunodetection of the production of the P. hemolytic leukotoxin ws done by the method of Hwkes et l. (9) with ntiser from rbbits immunized with gel-purified 105K ntigen (see below) nd from cttle experimentlly infected with P. hemolytic. Positive clones were cultured in LB-Amp nd checked for the production of the 105K ntigen by Western blotting nd immunoprecipittion. To test for toxin ctivity in the clones, 25-ml cultures grown in LB-Amp were concentrted fivefold in growth medium by centrifugtion nd lysed by single pssge through French press t 16,000 lb/in2. The lyste ws clered of unlysed cells by centrifugtion t 3,000 x g for 5 min nd used in seril dilutions in the BL-3 ssy (see bove). RESULTS 105K polypeptide toxin component. The superntnt fluids of P. hemolytic biotype A, serotype 1 cultures grown in either of two commonly used growth medi, BHI or DMEM- FCS, hd comprble levels of leukotoxin ctivity (200 U/ml for DMEM nd for 400 U/ml for BHI) s judged by the BL-3 cytotoxicity ssy. This toxin ctivity ws inctivted by ntiserum obtined from cttle infected with live P. hemolytic cells (Tble 1). Immunoprecipittion with this ntiserum of the culture superntnts from number of clinicl isoltes of P. hemolytic biotype A, serotype 1, which is most commonly ssocited with pneumonic psteurellosis, nd from other serotypes reveled number of mjor bnds (Fig. 2). The mjor common feture ws the 105K species, which we provisionlly identified s component of the toxin. On the bsis of the bnd intensity in nlyses such s the one shown in Fig. 2, we estimte tht pproximtely 0.5,ug of 105K protein per ml ws present in superntnts from cultures in lte logrithmic phse. Initil ttempts to purify the toxin from BHI nd DMEM-FCS filed, becuse these medi contin grm quntities of nondilyzble, mmoniumsulfte-precipitble mteril. Insted, it ws found tht DBHI cn serve s n cceptble growth medium, typiclly yielding 1,000 U of toxin ctivity per ml in the lte log phse (Fig. 3A). Overnight ertion of the sturted culture resulted in the loss of the toxin ctivity, s reported previously (2). Immunoprecipittion with the bovine ntibody shows tht the kinetics of the ccumultion of the 105K species prlleled the time course of toxin production (Fig. 3B). Accompnying the loss of toxin ctivity during the overnight ertion ws the degrdtion of the 105K species into lower-moleculr-weight immunorective forms (Fig. 3B, lne 10). Proteolytic degrdtion of the 105K species ws lso seen in BHI nd DMEM-FCS, with the principl degrded form 95K species (dt not shown). Toxin neutrliztion by nti-105k ntibodies. Antibodies were rised in rbbits ginst the 105K polypeptide purified by preprtive SDS-PAGE. These ntibodies neutrlized toxin ctivity (Tble 1) nd showed single precipitin line in Ouchterlony immunodiffusion gels (dt not shown). Western blots using this ntiserum demonstrted not only tht single superntnt bnd ws lbeled, but lso tht the cell pellet contined nother immunorective species t 95 kilodltons (Fig. 4). Chromtogrphic behvior of toxin. Superntnt from lte log-phse culture ws subjected to severl kinds of chromtogrphic nlysis. Toxin ctivity eluted from DEAE-cellulose t 0.3 to 0.4 M NCl (Fig. 5A), nd SDS- PAGE nlysis of the column frctions reveled tht the 105K species ws lso eluted in the sme frctions (Fig. SB).

4 VOL. 55, 1987 PASTEURELLA HAEMOLYTICA LEUKOTOXIN 2351 AT Ss tm t i 0 ) C-, 5- S6 -r1000 m + C C t500 1 * --I /N Time (min.) B FIG. 4. Western blot nlysis by using the nti-105k ntibody. The rbbit ntiserum prepred ginst the gel-purified 105K species ws used t 1:200 dilution. Lnes: 1, DBHI control; 2, cellulr proteins; 3, culture superntnt l-w --- :--W ii*- ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. 68-4ț ~ ~ ~~~~~~~~~~~~~ I s s i FIG. 3. Kinetics of leukotoxin production nd the ppernce of the 105K species during culture growth. P. hemolytic cells were grown in DBHI, nd smples were removed t vrious times. The smples were ssyed for leukotoxin ctivity nd for the presence of the 105K polypeptide s described in Mterils nd Methods. (A) Kinetics of culture growth nd leukotoxin production. (B) Kinetics of 105K polypeptide synthesis. Lnes: 1, moleculr weight stndrds; 2 to 11, immunoprecipittion by using 10,ul of the bovine ntiserum nd 300 RlI of DBHI (lne 2) or 300,ul of culture superntnt from smples tken from culture t 180 min (lne 3), 210 min (lne 4), 240 min (lne 5), 270 min (lne 6), 300 min (lne 8), 330 min (lne 9), 360 min (lne 10), nd 24 h (lne 11). Neither toxin ctivity nor the 105K species ws bound to crboxymethyl-cellulose t ph 6.5 or to octyl-sephrose t ph 8.0 in 1 M (NH4)2S04 (dt not shown). In ddition, the 105K polypeptide nd toxin ctivity showed pprent moleculr weights greter thn 400,000 bsed on gel filtrtion chromtogrphy (Fig. 6). Identifiction of cloned toxin gene product. Plsmid librries of P. hemolytic genomic DNA were screened with the bovine nd rbbit ntiser, nd one clone, contining the plsmid pyfc19 with 3.9-kilobse-pir insert, ws shown to rect with both ntiser nd to produce intrcellulr toxin ctivity (pproximtely 50 U/mI of overnight culture [dt not shown]). A 105K polypeptide could be immunoprecipitted from lystes of the clone. In ddition, lower-moleculr-weight species, especilly 95K polypeptide, were immunoprecipitted t vrious proportions of the 105K polypeptide, depending on the mnner of cell disruption 2 cir 0 A ± C 1.OM * #' s _~ 13 PI 0.5M- ~500 _ 4~ ~ ~ ~~ 0) Frction B c 68 -b Qt--; _.. FIG. 5. Elution of leukotoxin ctivity nd the 105K species from DEAE-cellulose. Crude leukotoxin prepred s described in Mterils nd Methods ws pplied to DEAE-cellulose column (1 by 8 cm) in 20 mm Tris, ph 8.0. Elution ws ccomplished with liner slt grdient (O to 0.7 M NCl). (A) Ech frction (6 ml) ws ssyed for toxin ctivity by seril dilution. Approximtely 70o of the pplied toxin ctivity ws recovered. (B) Smples from selected frctions were nlyzed by SDS-PAGE. Lnes 1 nd 12 contin moleculr weight stndrds. Lnes 2 to 11 contin frctions 11 to 20, respectively E 4 E

5 2352 CHANG ET AL. INFECT. IMMUN. ^ t I m I 'E co c x 0 --I kbp C PV1 UH,.C PV HU 3.9kbp ' 1 3.9kbp FIG. 8. Comprison of restriction mps of P. hemolytic DNA inserts in pyfc19 nd pph5. The mp of pph5 is from Lo et l. (13). The discrepncy between the plcement of the PvuI site in the two mps is unexplined. Abbrevitions: A, AvI; C, ClI; H, HindIll; P, PstI; U, PvuI; V, PvuII; kbp, kilobse pirs. A A & Frction FIG. 6. Gel permetion chromtogrphy of the leukotoxin. Crude leukotoxin prepred s described in Mterils nd Methods ws pplied to Sephcryl S-300 column (2.5 by 45 cm) nd eluted with 20 mm Tris, ph 8.0. Ech frction (2.5 ml) ws ssyed for leukotoxin ctivity by seril dilution. Approximtely 60% of the pplied leukotoxin ctivity ws recovered. Frctions 41 to 110 contined less thn 50 U of leukotoxin ctivity per ml. OD280, Opticl density t 280 nm; V, void volume;,8-gl, (3-glctosidse; BSA, bovine serum lbumin. (Fig. 7). Restriction nlysis of the insert showed tht the cloned frgment hd significnt overlp with the 6.3-kilobse frgment reported by Lo et l. (13) to encode toxin ctivity (Fig. 8). Locliztion of cross-recting mteril to the cell surfce. Since the nti-105k ntibody rected with 95K species in the cell pellet (Fig. 4), the cell surfce ws exmined for evidence of cross-recting mteril by indirect immunofluorescence. While the ntibody ginst Formlin-fixed cells lbeled the surfce of P. hemolytic uniformly, the nti- 105K ntibody gve nonuniform or punctte lbeling pttern (Fig. 9). This punctte distribution ws not reproduced by using the preimmune serum nd ws not ffected by lbeling t low temperture or with monovlent Fb frction (dt not shown). DISCUSSION We identified 105K polypeptide s the sole polypeptide component of the leukotoxin produced by P. hemolytic. This moleculr species ws preliminrily identified s cndidte for the toxin bsed on the observtion tht the :: FIG. 7. Immunoprecipittion of cloned leukotoxin produced in E. coli. Inimunoprecipittions were done with 10,u1 of the bovine ntiserum nd 300 p.l of P. hemolytic culture superntnt (lne 1), 20,ul of French-pressed cell lystes from cultures grown in LB-Amp (lnes 2 nd 3), or whole-cell extrcts from cultures grown in miniml glycerol medium (lnes 4 nd 5) prepred s described in Mterils nd Methods. Lnes: 2 nd 4, E. coli TBlpHG165; 3 nd 5, E. coli TBlpYFC19. Moleculr sizes re shown in kilodltons. kinetics of its ppernce nd disppernce from the superntnts of P. hemolytic cultures prlleled those of the cytotoxin ctivity. In ddition, secretion of the 105K species ws common feture of ll pthogenic serotypes. Antibodies rised ginst the 105K polypeptide were shown to neutrlize toxin ctivity. Previous reports hve indicted tht the cytotoxin is het-lbile protein of moleculr weight of 150,000 or greter s judged by gel filtrtion (10) nd ultrfiltrtion (2). These results cn be reconciled with our present findings by ssuming tht the ctive toxin is multimer of the 105K protein identified on denturing gels. In fct, our preliminry ttempts to purify the toxin by gel filtrtion suggest tht its moleculr weight is much greter thn 400,000 (Fig. 6). More difficult to reconcile is the clim tht the cytotoxin is composed of 20K nd 50K subunits (10). This pprent contrdiction might be due to the fct tht cytotoxin ctivity is unstble (Fig. 1) nd tht the 105K protein we identified s the toxin is subject to proteolytic degrdtion. Previous reports hve indicted tht there is n extrcellulr protese relesed by P. hemolytic nd lso tht the cells utolyse in sttionry phse (6), potentilly relesing intrcellulr proteolytic ctivities. In fct, the studies purporting to demonstrte the existence of 20K nd 50K subunits were performed by using preprtions derived from culture superntnts hving vrible mounts of lowmoleculr-weight mteril which ws ntigeniclly similr to the high-moleculr-weight (150K) form of the toxin (10). In our hnds, the primry brekdown product in culture superntnts ws 95K polypeptide (Fig. 3B), lthough subsequent degrdtion to lower-moleculr-weight immunorective forms hs lso been seen (dt not shown). Our dt support the ide tht the 105K species is the only protein component of the ctive toxin. The size of the cloned P. hemolytic DNA frgment which encodes the toxin protein is only bout 0.9 kilobse pirs lrger thn the minimum required for polypeptide of this size. Nevertheless, the toxin synthesized in E. coli ws pprently full length nd fully ctive, indicting tht no modifiction specific to P. hemolytic ws required. In ddition, the most highly purified frctions from ion-exchnge chromtogrphy show only the 105K species nd the 95K brekdown product (Fig. SB). When whole-cell extrcts of P. hemolytic were subjected to Western blot nlysis by using the 105K-specific ntibody, 95K bnd ws lbeled in ddition to the 105K species. It is not cler wht reltionship this species hs to the ctive secreted toxin. At lest one of these two forms is locted t the cell surfce, s judged by indirect immunofluorescence, nd is distributed in nonuniform, punctte pttern. It is possible this punctte distribution reflects structurl fetures of the P. hemolytic envelope which re involved in the secretion of the extrcellulr toxin nd my be similr to the zones of dhesion reported by Byer (3).

6 VOL. 55, 1987 PASTEURELLA HAEMOLYTICA LEUKOTOXIN 2353 FIG. 9. Indirect immunofluorescence nlysis of P. hemolytic surfce ntigens. P. hemolytic cells were stined nd immobilized for microscopy s described in Mterils nd Methods. Fluorescence microgrphs of cells lbeled with bovine ntiserum (A) or rbbit nti-105k ntiserum (B) s the first ntibody. (C nd D) Phse-contrst microgrphs of the sme fields s shown in pnels A nd B, respectively. Mgnifiction, x780. ACKNOWLEDGMENTS We re grteful to Terrn Moulds nd Jeff Smith for technicl ssistnce, to Lis Lohmn for her skilled grphics, to Lydi McCrtney for dministrtive ssistnce, nd to the Biocommunictions Office of the College of Medicine, Texs A&M University, for photogrphic services. We re indebted to D. Kremer nd R. Lon of the College of Veterinry Medicine, Texs A&M University, for ccess to cttle nd serum of infected cttle, respectively. This work ws supported by Texs Advnced Technology Reserch Progrm grnt to R.Y. nd D.S. LITERATURE CITED 1.Altmn, E., R. K. Altmn, J. M. Grrett, R. J. Grimil, nd R. Young S gene product: identifiction nd membrne locliztion of lysis control protein. J. Bcteriol. 155: Bluyut C. S., R. R. Simonson, W. J. Bemrick, nd S. K. Mheswrn Interction of Psteurell hemolytic with bovine neutrophils: identifiction nd prtil chrcteriztion of cytotoxin. Am. J. Vet. Res. 42: Byer, M. E The fusion sites between outer membrne nd cytoplsmic membrne of bcteri: their role in membrne ssembly nd virus infection, p In M. Inouye (ed.), Bcteril outer membrnes: biogenesis nd function. John Wi- ley & Sons, New York. 4. Chng, Y. F., nd H. W. Renshw Psteurell hemolytic leukotoxin: comprison of 51Cr-relese, trypn blue dye exclusion, nd luminol-dependent chemiluminescence-inhibition ssys for sensitivity in detecting leukotoxin ctivity. Am. J. Vet. Res. 47: Chng, Y. F., H. W. Renshw, R. J. Mrtens, nd C. W. Livingston, Jr Psteurell hemolytic leukotoxin: chemiluminescent responses of peripherl blood leukocytes from severl different mmmlin species to leukotoxin- nd opsonintreted living nd killed Psteurell hemolytic nd Stphylococcus ureus. Am. J. Vet. Res. 47: Dtulbowski, G. L., P. E. Shewen, A. E. Udoh, A. Mellors, nd B. N. Wilkie Proteolysis of siloglycoprotein by Psteurell hemolytic culture superntnt. Infect. Immun. 42: Frnk, G. H., nd G. E. Wessmn Rpid plte gglutintion procedure for serotyping Psteurell hemolytic. J. Clin. Microbiol. 7: Greer, C. N., nd P. E. Shewen Automted colorimetric ssy for the detection of P. hemolytic leukotoxin. Vet. Microbiol. 12: Hwkes, R., E. Nidy, nd J. Gordon A dotimmunobinding ssy for monoclonl nd other ntibodies. Anl. Biochem. 119:

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