Vol. 61, No. 11INFECTION AND IMMUNITY, Nov. 1993, p. 4682-46880019-9567/93/114682-07$02.00/0Copyright X 1993, American Society for Microbiology

Three Contiguous Lipoprotein Genes in Pasteurellahaemolytica Al Which Are Homologous to a Lipoprotein

Gene in Haemophilus influenzae Type bBRAD J. COONEY AND REGGIE Y. C. LO*

Department ofMicrobiology and The Canadian Bacterial Diseases Network,University of Guelph, Guelph, Ontario, Canada NJG 2W1

Received 20 May 1993/Returned for modification 21 July 1993/Accepted 26 August 1993

An Escherichia coli clone carrying the recombinant plasmid pPH24 has been found to express highlyimmunoreactive antigens of PasteureUla haemolytica Al. Two or three antigens of approximately 30 kDa werelocated to both the inner and outer membranes of the E. coli clone and in P. haemolytica Al. From the insertDNA of 8.2 kbp on pPH24, a fragment of 4.6 kbp was found to code for these antigens. Nucleotide sequenceanalysis of the 4.6-kbp DNA identified three genes (designatedplpA, -B, and -C) arranged in tandem which codefor three proteins, Plpl, -2, and -3, with predicted molecular masses of 30.1, 30.3, and 29.0 kDa, respectively.Comparison of the nucleic acid sequence of plpA, -B, and -C with GenBank sequences showed extensivehomology with a Haemophilus influenzae 28-kDa lipoprotein gene. [14Cjpalmitate labelling coupled withglybomycin inhibition experiments showed that Plpl, -2, and -3 are also lipoproteins. In addition, plpA, -B, and-C were found to be present only in A biotypes ofP. haemolytica by Southern blot analysis. Since Pip], -2, and-3 were found to be antigenic components in a culture supernatant vaccine, they could be candidates for furtherinvestigation as vaccine components.

Pasteurella haemolytica Al is the principle microorgan-ism associated with bovine pneumonic pasteurellosis, amajor cause of sickness and economic loss in the feedlotindustry in North America (21, 35). Numerous attempts havebeen made by various researchers to develop an effectivevaccine against P. haemolytica Al, most with limited suc-cess (4, 6, 20). Recently, a concentrated cell-free culturesupernatant vaccine, Presponse, which shows considerableefficacy in both experimental and field trials, has beendeveloped (27). However, the important protective antigensin Presponse have not been defined.

Since Presponse is primarily a concentrated culture super-natant, it contains molecules secreted by P. haemolytica Alas well as surface molecules released or sloughed off duringcell division. Some of these antigens include secreted pro-teins such as a leukotoxin, a glycoprotease, and a neuramin-idase as well as membrane proteins, capsule, and lipopoly-saccharide materials (1, 2, 8, 11, 32). One of the importantantigens in this collection is the leukotoxin (Lkt), a cytolysinspecific for ruminant leukocytes (14, 28). The genetic deter-minant which encodes Lkt has been cloned and character-ized in several laboratories (13, 17, 18, 31). In our labora-tory, the leukotoxin has been expressed in high levels inEscherichia coli after genetic manipulation of the lkt deter-minant (30). Supplementation of recombinant Lkt to Pre-sponse increased its efficacy (9), demonstrating the utility ofsuch an approach in the development of a more efficaciousvaccine.

In our continuing efforts to characterize the culture super-natant (soluble) antigens of P. haemolytica Al, we haveisolated a collection of recombinant plasmids coding forthese antigens (16). We report here the characterization ofthe genes which encode three lipoproteins of P. haemolyticaAl present in the culture supernatant.

* Corresponding author.

MATERIALS AND METHODS

Bacteria, plasmids, and culture conditions. The recombi-nant plasmid pPH24 was initially isolated among the collec-tion of recombinant E. coli clones expressing P. haemolyticaAl soluble antigens (16). E. coli HB101 and TG-1, plasmidpBR322, and the M13 phage vectors mpl8 and mpl9 weredescribed previously (16, 17, 18). P. haemolytica Al wasgrown on brain heart infusion broth, E. coli HB101 cloneswere grown on LT supplemented with ampicillin at 100mg/liter, and E. coli TG-1 was grown on Davis minimalmedia (16).Enzymes and chemicals. Restriction endonucleases and

DNA modifying enzymes were purchased from BethesdaResearch (Burlington, Ontario, Canada) or PharmaciaChemicals, Inc. (Dorval, Quebec, Canada), and used asdescribed by the suppliers. Radioisotopes were purchasedfrom ICN Biomedical (Montreal, Quebec, Canada) or Am-ersham Laboratories (Oakville, Ontario, Canada). Goat anti-rabbit or anti-calf immunoglobulin G-alkaline phosphataseconjugate and immunodetection reagents were purchasedfrom Bio-Rad Laboratories (Mississauga, Ontario, Canada).

Analysis of membrane fractions. Outer and inner mem-branes were prepared by sucrose gradient centrifugation,and their purity was assayed as described previously (19).The membranes were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as de-scribed by Laemmli (15). Typically, the stacking gel con-sisted of 4% acrylamide, and the separating gel consisted of15% acrylamide. After electrophoresis, the proteins werevisualized by staining with Coomassie blue R250 or detectedwith antibodies in Western blot (immunoblot) analysis.For Western blot analysis, the proteins were transferred

onto nitrocellulose as described previously (5). The firstantibody used was either a rabbit antiserum raised againstthe soluble antigens of P. haemolytica Al or a serum from acalf vaccinated with Presponse. An appropriate secondantibody (either goat anti-rabbit or anti-calf) conjugated to

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THREE LIPOPROTEIN GENES IN P. HAEMOLYTICA Al 4683

alkaline phosphatase was used to detect the bound firstantibodies. The color development and reagents used weredescribed previously (17).Recombinant DNA techniques. Subclones of pPH24 were

made by digestion and religation of appropriate plasmidDNA fragments by using available restriction sites. Tosequence the insert DNA, the DNA fragments were purifiedafter low melting-point agarose gel electrophoresis withGene Clean reagents (BioCan, Mississauga, Ontario, Can-ada) and cloned into appropriately digested M13 mp18 ormpl9 double-stranded DNA. Both strands of the DNA weresequenced by the dideoxy chain termination method asdescribed previously (18, 31) with a T7 sequencing kit fromPharmacia. Specific oligonucleotide primers were synthe-sized with an Applied Biosystem International model 391EPunit. The nucleotide sequences were compiled and analyzedby using the Pustell programs (IBI, Toronto, Ontario, Can-ada) or the PC-Gene programs. The nucleotide sequence wasalso used to search for homology against the GenBank database.Agarose gel electrophoresis and Southern blot analysis.

Southern blot analysis was carried out as described previ-ously (17). Briefly, chromosomal DNA from the referencestrains of the 16 serotypes of P. haemolytica were digestedwith the restriction enzyme ClaI, and the DNA fragmentswere separated on a 0.7% agarose gel and electrophoreticallytransferred onto nitrocellulose paper. The probe used was arestriction fragment from pPH24 containing the coding re-gions of the plp genes. The probe was labelled with a [32P]dATP by nick translation and hybridized as described pre-viously (17).

Labelling of proteins with [14CJpalmitate. Proteins werelabelled as described by Theisen et al. (33). Briefly, E. coliHB101/pPH24 was grown in LT with ampicillin supple-mented with glycerol (0.5% [wtlvoll) and Casamino Acids(2% [wt/vol]). The cells were grown to log phase to which 10pCi of [14C]palmitate (specific activity, 827 mCi/mmol) wasadded and incubated for 2 h. Labelling of the proteins wasterminated by the addition of trichloroacetic acid to 10%(final concentration) for 30 min on ice. Proteins were pelletedat 15,000 x g for 20 min, and the pellets were washed twicewith methanol. The dried pellets were resuspended in 200 ,ulof sample buffer and analyzed by SDS-PAGE. The radiola-belled proteins were detected by autoradiography by usingAmplify (Amersham) to enhance the signals. For the inhibi-tion of signal peptidase II activity, globomycin (10 mg/ml indimethyl sulfoxide) was added to a final concentration of 100,ug/ml for 5 min prior to the addition of ['4C]palmitate.

Nucleotide sequence accession number. The nucleotidesequence of the 4.6-kbp DNA fragment containingplpA, -B,and -C has been deposited in the GenBank data base underthe accession number L16627.

RESULTS

Isolation and characterization of pPH24. E. coli HB101carrying pPH24 consistently exhibited a very strong anti-genic reaction with the rabbit antiserum raised against thesoluble antigens of P. haemolytica Al. To determine thenature and localization of this antigenic response, outer andinner membrane fractions of E. coli/pPH24 were preparedand analyzed by Western blot. The results in Fig. 1A showthat two, perhaps three, immunoreactive proteins with mo-lecular masses of approximately 30 kDa could be identifiedin both membrane fractions ofE. coli/pPH24. Similarly sizedimmunoreactive proteins could also be seen in both mem-

AM 1 2 3 4 5 6

EU;_

43-

29-

BM 1 2 3 4 5 6

43-

.429-

FIG. 1. Western blot of inner and outer membranes from P.haemolytica Al, E. coli/pBR322, and E. coli/pPH24. Inner (lanes 1,3, and 5) and outer (lanes 2, 4, and 6) membrane fractions of E.coli/pBR322, P. haemolytica Al, and E. coli/pPH24, respectively,were used. The first antiserum used in blot A was the rabbitantiserum raised against the culture supernatant (soluble antigens)of P. haemolytica Al. The first antiserum used in blot B was anantiserum from cattle vaccinated with the culture supernatant Pre-sponse vaccine. Lane M, molecular size standards in kilodaltons.The arrows indicate the positions of the immunoreactive proteins ofinterest.

brane fractions from P. haemolytica Al. To determine ifthese proteins were present in the culture supernatant vac-cine Presponse, serum from a calf vaccinated with Pre-sponse was used in the Western blot analysis. The results inFig. 1B showed that the Presponse vaccine stimulated theproduction of antibodies against these proteins, suggestingthat they may be important antigenic components in Pre-sponse.The insert DNA on pPH24 was mapped to be 8.2-kbp long.

A number of subclones were made and tested for theexpression of the 30-kDa proteins. The results showed thatthe proteins were encoded on a DNA fragment of approxi-mately 4.6 kbp to the left of the PvuII site (Fig. 2). Withoutfurther suitable restriction sites for subcloning, and sinceapproximately 3 kbp of DNA would be necessary to encodethe three proteins separately, the nucleotide sequence of thisentire 4.6 kbp was determined.

Nucleotide sequence analysis. The DNA between the PvuIIsite and the Sau3A/BamHI junction contained 4,609 bp. Thesequence is numbered from the PvuII site as position 1;however, only the pertinent sequences from 1,600 to 4,609

(A 2

C B.p

G

2 kbp

A 30kDaantigens

P Pl2 2 1----- +B r.e A A

A

+

FIG. 2. Restriction endonuclease map of plasmid pPH24. Sym-bols: EJ, pBR322 presented linearly at the coordinate of 3 kbp; A,DNA deleted in three subclones; + or -, presence or absence,respectively, of the 30-kDa antigens as demonstrated by Westernblot. The positions and orientations ofplpA, -B, and -C are indicatedby the dark arrows. Abbreviations: A, AvaI; B, BclI; C, ClaI; P,PstI; P2. PvuII.

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FIG. 3. Nucleotide sequence of the pertinent part of the insert DNA of pPH24 and deduced amino acid sequence of the P. haemolyticaAl lipoproteins Plpl, -2, and -3. Potential ribosome binding sites (SD) and restriction endonuclease sites are indicated. The putativerho-independent termination signal is indicated by the inverted arrows. Abbreviations: P, PstI; B, Bcll.

are presented in Fig. 3. When the entire nucleotide sequencewas analyzed, it was found to contain three open readingframes arranged in tandem (Fig. 2 and 3). Each open readingframe is preceded by a sequence that could be transcribedinto a ribosome binding site. The number of open readingframes detected correlates to the number of immunoreactiveproteins expressed from E. coli/pPH24. Even though there isno evidence that the three reading frames are expressed as

an operon, their close proximity suggests that it is likely thatthey are coordinately expressed. The position of a promotercould not be conclusively identified without primer exten-sion analysis; however, a sequence which could function asa rho-independent termination signal was present after thethird open reading frame. The three open reading frameswere designatedplpA, -B, and -C in order of their positions.The predicted molecular weight of the proteins that would

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THREE LIPOPROTEIN GENES IN P. HAEMOLYTICA Al 4685

....Plpl NSFKKILGVA LV----SAIA LTACKWZA ZSTRAPAAQA ----PAXIKV 42Plp2 NNFKKLLGVA LV----SAIA LTACE-KA ---APATTA KTXNKAPLKV 42Plp3 NKINKLAGAV AIF---S-L LTAD--KA --------- ------KLKV 29Hilp N---KLKQLF AITAXASALV LTGKDP AAAAPL--- ------KIKV 38

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Plpl VDTSIAAUVS --DVDLAVVW NIVAG-QVGL N-TQOHGVFV 3SDPVNI 237

Pip2 ADTS-LLTR LIOVZAVIM TAG-GL 8-PDIIDGXV KSDSPYVNL 23sPlp3 IEAP-LLPRT LDOVAFSIIU TTYAGXTV-K AMQS--GXVV ZDKDSPYVL 221Hilp VDTSVAA-R& LDDVDIAVVN NTYAG-QYOL NA-QDGV WKDSPVNI 232

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.^ * .^.. 4.... ^^^^*...4 . ..^^

FIG. 4. Amino acid alignment of Plpl, -2, and -3 of P. haemolyt-ica Al and the 28-kDa lipoprotein from H. influenzae type b (Hilp).The position of the signal sequence for signal peptidase II and theinvariant cysteine residue are indicated by the arrows. An asteriskdenotes amino acids present in all four proteins: a period indicatesamino acids present in three of the four proteins; dashes representgaps introduced to maximize alignment. The numbers to the rightindicate the positions of the amino acids in the protein.

be expressed fromplpA, -B, and -C were 30.1, 30.3, and 29.0kDa, respectively. The corresponding proteins were desig-nated Plpl, -2, and -3, respectively. The molecular weightsof Plpl, -2, and -3 are very similar to the sizes of the proteinsdetected in the Western blot analysis expressed from the E.coli clone and from P. haemolytica Al, and it is presumedthat each corresponds to one of the three proteins. Sincethese proteins are so similar in size, it is not unexpected thatthey do not resolve into distinct bands upon SDS-PAGEanalysis.Nature of the recombinant proteins. A homology search of

the entire 4,609-bp sequence against the GenBank data baseidentified extensive homology with a gene coding for a28-kDa lipoprotein from Haemophilus influenzae type b (7).It is apparent thatplpA, -B, and -C represent three copies ofa gene homologous to the H. influenzae 28-kDa lipoproteingene. On the basis of the nucleotide sequence comparison,plpA is the most similar of the three to the H. influenzae28-kDa lipoprotein gene. To a lesser extent, homology wasalso detected with an E. coli 28-kDa inner membrane li-poprotein (36) during the search (data not shown).A comparison of the predicted amino acid sequences of

Plpl, -2, -3, and the H. influenzae 28-kDa lipoprotein isshown in Fig. 4. It could be seen that Plpl, -2, and -3 allpossess features similar to the H. influenzae 28-kDa lipopro-tein at the N terminus which are characteristic of lipopro-teins. It is evident that Plpl, -2, and -3 must also belipoproteins. Throughout the entire amino acid sequences,there are remarkable identities and similarities among thefour proteins, with the degree of homology ranging from 66to 83%. A similar comparison between the H. influenzae28-kDa lipoprotein and the E. coli 28-kDa inner membranelipoprotein has been reported by Yu et al. (36). A dendro-gram of the relatedness between all five lipoproteins isshown in Fig. 5.

Palmitate labelling and globomycin inhibition. The similar-

50 60 70 80 90 160

FIG. 5. Dendrogram showing genetic relationship between thelipoproteins Plpl, -2, and -3 of P. haemolytica Al, the 28-kDalipoprotein from H. influenzae type b (Hilp), and the 28-kDalipoprotein from E. coli (Eclp). The horizontal axis shows percentsimilarities between the proteins.

ity of Plpl, -2, and -3 to the H. influenzae 28-kDa lipoproteinand the presence of a possible cleavage site for signalpeptidase II lead one to conclude that they are lipoproteins.To support this hypothesis, palmitate labelling and globomy-cin inhibition experiments were carried out. Figure 6 showedthat the recombinant lipoproteins from E. coli/pPH24 werelabelled with [14C]palmitate and showed a higher molecularweight when the cells were treated with globomycin duringthe labelling period.

Southern blot analysis. Chromosomal DNA from the 16serotypes of P. haemolytica was digested with ClaI andprobed with the insert DNA carrying pipA, -B, and -C.Figure 7 shows that the A biotypes (serotypes 1, 2, 5, 6, 7, 8,9, 11, 12, 13, 14, and 16) have homology to the probewhereas the T biotypes (serotypes 3, 4, 10, and 15) showlittle or no hybridization with the probe. These results alsoshow that the probe hybridized to different-sized DNAfragments among some of theA serotypes. This could be dueto the differences in positions of the flanking ClaI sites or thenumbers of copies of the plp genes in the different isolates(see below).

DISCUSSION

From the collection of recombinant clones expressing P.haemolytica Al antigens, we have identified and character-ized the genes coding for the leukotoxin (17, 18, 31), aserotype 1-specific outer membrane protein (19), and theglycoprotease (1). Because of the very strongly antigenicnature of E. coli/pPH24, we anticipated it encoded a highlyimmunoreactive antigen(s). Further, since the initial rabbitantiserum was raised against culture supernatant antigens,we expect the antigen(s) is likely secreted or outer mem-brane molecules. Therefore, it was unexpected to observethe localization of Plpl, -2, and -3 in both the inner and outermembranes in the E. coli clone as well as in P. haemolyticaAl. Nakai and Kanehisa (25) examined the amino acidsequences of various proteins and devised a scheme todetermine their cellular location. According to their scheme,if the second or third amino acid of a mature lipoprotein isnegatively charged, then the lipoprotein is localized to theinner membrane. Examination of the predicted amino acidsequences of Plpl, -2, and -3 of P. haemolytica Al by usingthis scheme suggest that all three lipoproteins should belocalized to the inner membrane. It is also possible that theproteins are anchored to the inner membrane, span theperiplasmic space, and interact with components of the outer

PIPI

Hilp

PIp2

I PIp3

CID

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1 2 M.. ...............

R2:........

m[ | --29

FIG. 6. ["4C]palmitate labelling of lipoproteins from E. coli car-rying pPH24. An autoradiograph of cells labelled with [14C]palmitatein the presence of globomycin (lane 1) and cells labelled in theabsence of globomycin (lane 2) is shown. The locations of themature (m) and precursor (p) forms of the lipoproteins are indicated.M indicates the position of the molecular marker at 29 kDa.

membrane and that some materials are included in the outermembrane fraction during sample preparation. Further, hy-dropathic analysis of these proteins did not detect anyextensive transmembrane domains, consistent with the in-terpretation that they are not integral membrane proteins.Initially, it was expected that Plpl, -2, and -3 would besurface exposed since they were recognized by the antiseraraised against supernatant antigens. However, it is possiblethat they are released into the culture supernatant as a resultof cell lysis or membrane turnover and stimulate an antibodyresponse because of their highly immunogenic nature.Upon sequence analysis of the cloned DNA, it was

surprising to observe three genes arranged in tandem. Thepossibility that the presence of three genes could be due tocloning artifacts could be ruled out because although thethree genes are similar, they are only 66% identical innucleotide sequence. Further, polymerase chain reactionanalysis of the genomic DNA of P. haemolytica Al usingspecific primers flanking the three genes amplified a 2.7-kbpproduct as expected (unpublished result). The similarity ofthe predicted amino acid sequences of Plpl, -2, and -3 to the28-kDa lipoprotein of H. influenzae and the 28-kDa lipopro-tein of E. coli was another unexpected observation. Thishomology was observed throughout the entire protein, indi-

M 1 2 3 4 5 6 7 8 9 1011 1213141516.. .5.."'' ...

FIG. 7. Southern blot analysis showing the presence ofpipA, -B,and -C sequences in all A biotypes of P. haemolytica. Lanes 1 to 16,genomic DNA from P. haemolytica serotypes 1 to 16, respectively,digested with ClaI. Serotypes 1, 2, 5, 6, 7, 8, 9, 11, 12, 13, 14, and16 are all A biotypes which show hybridization with the pip probe.Lane M, X DNA digested with HindIII and hybridized with X DNAradiolabelled separately as size markers. The X size markers are 23,9.6, and 6.7 kbp from the top.

cating highly similar biological activities. The 28-kDa H.influenzae lipoprotein was suggested to be involved in trans-epithelial invasion ofH. influenzae type b strains, although itis not essential (7). No known function has been identifiedfor the E. coli 28-kDa inner membrane lipoprotein (36), but itis believed to be a minor membrane component.

Lipoproteins in general share a few common features suchas a short hydrophobic signal sequence which contains afour-amino-acid-sequence consensus cleavage site. The se-quence of the consensus cleavage site is L-X-Y-C, where Xand Y are small neutral amino acids (34). Upon removal ofthe signal sequence, the cysteine becomes the N-terminalresidue and is also the position of lipid modification. Thelipids are usually attached as one amide-linked and twoester-linked fatty acids (34). Examination of the predictedamino acid sequences of Plpl, -2, and -3 clearly indicated thepresence of the consensus cleavage and lipid modificationsequence L-T-A-C at the proper positions. Incidentally, thisconsensus sequence is also identical to that of a lipoproteinrecently characterized from the closely related bacteriumActinobacillus pleuropneumoniae (12). The lipoprotein na-ture of Plpl, -2, and -3 is demonstrated by the in vivolabelling of the recombinant proteins with palmitate in E. coli(Fig. 6). Furthermore, in the presence of glybomycin, cleav-age by signal peptidase II was inhibited and resulted in theaccumulation of precursor molecules. The highly antigenicnature of these recombinant proteins expressed from pPH24could be explained in part by their lipoprotein nature. It hasbeen well documented that lipoproteins are extremely im-munogenic because of their lipid moieties (3, 22), and thepresent data with both the rabbit and calf serum are consis-tent with this hypothesis. In addition, sera from colostrum-deprived calves do not contain antibodies to these lipopro-teins. However, antibodies against the lipoproteins appear 4weeks after vaccination with Presponse, and the titers cor-relate to resistance upon challenge, demonstrating that theselipoproteins are present in Presponse and stimulate an im-mune response (unpublished results). These lipoproteins arebeing further investigated as possible vaccine candidates.Mosier et al., by using sera from cattle vaccinated with

live or killed P. haemolytica Al vaccines against whole-cellantigens of P. haemolytica Al, identified a number ofantigens which are correlated with resistance to pneumonicpasteurellosis (23). In particular, an antigen of approx. 31kDa appeared to be very important. From their Westernblots, it appears there is more than one antigen in the 31-kDarange; in our current studies, we may have cloned the genescoding for some of these antigens. Craven et al. reported thecloning of a DNA fragment coding for a 30-kDa protein of P.haemolytica Al (10). The restriction map of their clonedDNA is completely different than that of the insert DNA onpPH24, and therefore it is unlikely that these are the sameDNA fragments. On the other hand, upon comparison of the4.6-kbp DNA sequence on pPH24 with the data base inGenBank, an entry containing the first copy of the lipopro-tein genes was retrieved although there was no associatedpublication (accession number M91072). It is not knownwhether this lipoprotein sequence entry is that determinedfrom the clones reported by Craven et al. (10), and if so, whythere is a discrepancy in the number of copies identified. It ispossible that the lipoprotein genes undergo genetic rear-rangements in different isolates of P. haemolytica Al as aresult of the presence of multiple copies. Therefore, theseresearchers may have isolated a DNA fragment from a P.haemolytica Al isolate which contains only a single copy ofthe lipoprotein gene.

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THREE LIPOPROTEIN GENES IN P. HAEMOLYTICA Al 4687

Data from Southern blot analysis showed that all Abiotype strains contain the plp genes while the T biotypestrains do not appear to contain these genes. These resultsare consistent with numerous data suggesting that the A andT biotypes of P. haemolytica are very different species andthat they should be classified into two separate species (24,26, 29). Regarding the number of the plp genes in the Abiotypes, it is possible that different A biotypes harbordifferent numbers of copies of these genes, similar to thesituation for the Al isolates.These results showed that three similar copies of lipopro-

tein genes are present in tandem on the genome of P.haemolytica Al and encode three proteins of very similarmolecular weights. At this time, it cannot be determinedwhich gene codes for which of the three proteins since theyare so similar in size. In addition, since Craven et al. (10)reported the isolation of a different DNA fragment coding for30-kDa P. haemolytica Al proteins, it is possible that thegenetic loci for these lipoproteins are variable within differ-ent P. haemolytica Al isolates. Further experiments will beconducted with a collection of P. haemolytica Al isolates as

well with other serotypes to address this issue.

ACKNOWLEDGMENTS

We thank Andy Potter for supplying the globomycin and assistingwith the GenBank search. We also thank Lori Burrows for editorialsuggestions.

This work was supported by a research grant from the CanadianBacterial Diseases Network (Networks of Centres of ExcellenceProgram).

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